Compositions and methods of use of targeting peptides for diagnosis and therapy of human cancer

ABSTRACT

The present invention concerns compositions comprising and methods of identification and use of targeting peptides selective for cancer tissue, particularly prostate or ovarian cancer tissue. The method may comprise identifying endogenous mimeotopes of such peptides, such as GRP78, IL-11Rα and hsp90. Antibodies against such targeting peptides or their mimeotopes may be used for detection, diagnosis and/or staging of prostate or ovarian cancer. In other embodiments, the compositions and methods concern a novel type of gene therapy vector, known as adeno-associated phage (AAP). AAP are of use for targeted delivery of therapeutic agents to particular tissues, organs or cell types, such as prostate or ovarian cancer. In still other embodiments, targeting peptides selective for low-grade lipomas may be used for detection, diagnosis and targeted delivery of therapeutic agents.

This application is a continuation in part of PCT patent applicationentitled, “Compositions and Methods of Use of Targeting Peptides AgainstPlacenta and Adipose Tissues,” by Pasqualini, Arap and Kolonin, filedAug. 30, 2002, which was a continuation in part of PCT/US01/27692, filedon Sep. 7, 2001. This application is also a continuation in part ofPCT/US01/28044, filed on Sep. 7, 2001 and of PCT/US01/27702, filed onSep. 7, 2001. The entire texts of all the above-cited applications areincorporated herein by reference.

This invention was made with U.S. government support under grants grantsCA90270, 1R1CA90810-01 and 1R01CA82976-01 from the National Institutesof Health. The U.S. government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention concerns the fields of cancer diagnostics andtargeted delivery of therapeutic agents to cancer cells. Morespecifically, the present invention relates to compositions and methodsfor identification and use of peptides that selectively target cancercell receptors, such as the IL-11 receptor and/or the GRP78 receptor. Inparticular embodiments, the targeted receptors are preferentiallyexpressed in prostate cancer, especially in metastatic prostate cancer.In certain embodiments, the invention concerns compositions and methodsof use of novel phage-based gene delivery vectors.

2. Description of Related Art

Therapeutic treatment of many disease states is limited by the systemictoxicity of the therapeutic agents used. Cancer therapeutic agents inparticular exhibit a very low therapeutic index, with rapidly growingnormal tissues such as skin and bone marrow affected at concentrationsof agent that are not much higher than the concentrations used to killtumor cells. Treatment of cancer and other organ, tissue or cell typeconfined disease states would be greatly facilitated by the developmentof compositions and methods for targeted delivery to a desired organ,tissue or cell type of a therapeutic agent.

Recently, an in vivo selection system was developed using phage displaylibraries to identify targeting peptides for various organs, tissues orcell types in a mouse model system. Phage display libraries expressingtransgenic peptides on the surface of bacteriophage were initiallydeveloped to map epitope binding sites of immunoglobulins (Smith andScott, 1986, 1993). Such libraries can be generated by inserting randomoligonucleotides into cDNAs encoding a phage surface protein, generatingcollections of phage particles displaying unique peptides in as many as10⁹ permutations. (Pasqualini and Ruoslahti, 1996, Arap et al, 1998a;Arap et al 1998b).

Intravenous administration of phage display libraries to mice wasfollowed by the recovery of phage from individual organs (Pasqualini andRuoslahti, 1996). Phage were recovered that were capable of selectivehoming to the vascular beds of different mouse organs, tissues or celltypes, based on the specific targeting peptide sequences expressed onthe outer surface of the phage (Pasqualini and Ruoslahti, 1996). Avariety of organ and tumor-homing peptides have been identified by thismethod (Rajotte et al., 1998, 1999; Koivunen et al., 1999a; Burg et al.,1999; Pasqualini, 1999). Each of those targeting peptides bound todifferent receptors that were selectively expressed on the vasculatureof the mouse target tissue (Pasqualini, 1999; Pasqualini et al., 2000;Folkman, 1995; Folkman 1997). Tumor-homing peptides bound to receptorsthat were upregulated in the tumor angiogenic vasculature of mice(Brooks et al., 1994b; Pasqualini et al., 2000). In addition toidentifying individual targeting peptides selective for an organ, tissueor cell type (Pasqualini and Ruoslahti, 1996; Arap et al, 1998a;Koivunen et al., 1999b), this system has been used to identifyendothelial cell surface markers that are expressed in mice in vivo(Rajotte and Ruoslahti, 1999).

This relative success notwithstanding, cell surface selection of phagelibraries has been plagued by technical difficulties. A high number ofnon-binder and non-specific binder clones are recovered using previousin vivo methods, particularly with components of the reticuloendothelialsystem such as spleen and liver. Removal of this background phagebinding by repeated washes is both labor-intensive and inefficient.Cells and potential ligands are frequently lost during the many washingsteps required. Methods that have been successful with animal modelsystems are unsatisfactory for identifying human targeting peptides,which may differ from those obtained in mouse or other animal modelsystems.

Attachment of therapeutic agents to targeting peptides has resulted inthe selective delivery of the agent to a desired organ, tissue or celltype in the mouse model system. Targeted delivery of chemotherapeuticagents and proapoptotic peptides to receptors located in tumorangiogenic vasculature resulted in a marked increase in therapeuticefficacy and a decrease in systemic toxicity in tumor-bearing mousemodels (Arap et al., 1998a, 1998b; Ellerby et al., 1999). However, thetargeted delivery of anti-cancer agents in humans has not yet beendemonstrated. The targeted receptors reported in previous studies may bepresent in angiogenic normal tissues as well as in tumor tissues and mayor may not be of use in distinguishing between normal tissues,non-metastatic cancers and metastatic cancer. A need exists for tumortargeting peptides that are selective against human cancers, as well asfor targeting peptides that can distinguish between metastatic andnon-metastatic human cancers.

Attempts have been made to target delivery of gene therapy vectors tospecific organs, tissues or cell types in vivo. Directing such vectorsto the site of interest would enhance therapeutic effects and diminishadverse systemic immunologic responses. Adenovirus type 5 (Ad5)-basedvectors have been commonly used for gene transfer studies (Weitzman etal., 1997; Zhang, 1999). The attachment of Ad5 to the target cell ismediated by the capsid's fiber knob region, which interacts with cellsurface receptors, including the coxsackie adenovirus receptor (CAR) andpossibly with MHC class I (Bergelson et al., 1997; Hong et al., 1997).Upon systemic administration in vivo, binding of virus to CAR can resultin unintended enrichment of vectors in non-targeted but CAR-expressingtissues. Conversely, target cells that express little or no CAR areinefficiently transduced. A need exists to develop novel gene therapyvectors to allow more selective delivery of gene therapy agents.

A need also exists to identify receptor-ligand pairs in organs, tissuesor cell types. Previous attempts to identify targeted receptors andligands binding to receptors have largely targeted a single ligand at atime for investigation. Identification of previously unknown receptorsand previously uncharacterized ligands has been a very slow andlaborious process. Such novel receptors and ligands may provide thebasis for new therapies for a variety of disease states, such as cancerand/or metastatic prostate cancer.

SUMMARY OF THE INVENTION

The present invention solves a long-standing need in the art byproviding compositions and methods of preparation and use of targetingpeptides that are selective and/or specific for human cancer tissues,such as metastatic prostate cancer. In some embodiments, the inventionconcerns particular targeting peptides selective or specific forprostate cancer, including but not limited to SEQ ID NO:5-35, SEQ IDNO:37, SEQ ID NO:39-67 and SEQ ID NO:83-129. Other embodiments concernsuch targeting peptides attached to therapeutic agents. In otherembodiments, cancer targeting peptides may be used to selectively orspecifically deliver therapeutic agents to target tissues, such asprostate cancer and/or metastatic prostate cancer. In certainembodiments, the subject methods concern the preparation andidentification of targeting peptides selective or specific for a giventarget cell, tissue or organ, such as prostate cancer.

One embodiment of the invention concerns isolated peptides of 100 aminoacids or less in size, comprising at least 3 contiguous amino acids of atargeting peptide sequence, selected from any of SEQ ID NO:5-35, SEQ IDNO:37, SEQ ID NO:39-67 and SEQ ID NO:83-129. In a preferred embodiment,the isolated peptide is 50 amino acids or less, more preferably 30 aminoacids or less, more preferably 20 amino acids or less, more preferably10 amino acids or less, or even more preferably 5 amino acids or less insize. In other preferred embodiments, the isolated peptide may compriseat least 4, 5, 6, 7, 8 or 9 contiguous amino acids of a targetingpeptide sequence, selected from any of SEQ ID NO:5-35, SEQ ID NO:37, SEQID NO:39-67 and SEQ ID NO:83-129.

In certain embodiments, the isolated peptide may be attached to amolecule. In preferred embodiments, the attachment is a covalentattachment. In various embodiments, the molecule is a drug, achemotherapeutic agent, a radioisotope, a pro-apoptosis agent, ananti-angiogenic agent, a hormone, a cytokine, a growth factor, acytotoxic agent, a peptide, a protein, an antibiotic, an antibody, a Fabfragment of an antibody, a survival factor, an anti-apoptotic factor, ahormone antagonist, an imaging agent, a nucleic acid or an antigen.Those molecules are representative only and virtually any molecule maybe attached to a targeting peptide and/or administered to a subjectwithin the scope of the invention. In preferred embodiments, thepro-aptoptosis agent is gramicidin, magainin, mellitin, defensin,cecropin, (KLAKLAK)₂ (SEQ ID NO:1), (KLAKKLA)₂ (SEQ ID NO:2), (KAAKKAA)₂(SEQ ID NO:3) or (KLGKKLG)₃ (SEQ ID NO:4). In other preferredembodiments, the anti-angiogenic agent is angiostatin5, pigmentepithelium-derived factor, angiotensin, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin 12, platelet factor 4, IP-10,Gro-β, thrombospondin, 2-methoxyoestradiol, proliferin-related protein,carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate,angiopoietin 2 (Regeneron), interferon-alpha, herbimycin A, PNU145156E,16K prolactin fragment, Linomide, thalidomide, pentoxifylline,genistein, TNP-470, endostatin, paclitaxel, docetaxel, polyamines, aproteasome inhibitor, a kinase inhibitor, a signaling inhibitor (SU5416,SU6668, Sugen, South San Francisco, Calif.), accutin, cidofovir,vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline. Infurther preferred embodiments, the cytokine is interleukin 1 (IL-1),IL-2, IL-5, IL-10, IL-11, IL-12, IL-18, interferon-γ (IF-γ), IF-α, IF-β,tumor necrosis factor-α (TNF-α), or GM-CSF (granulocyte macrophagecolony stimulating factor). Such examples are representative only andare not intended to exclude other pro-apoptosis agents, anti-angiogenicagents or cytokines known in the art.

In various embodiments, targeting peptides attached to one or moretherapeutic agents may be administered to a subject, such as a humansubject. Such administration may be of use for the treatment of variousdisease states, such as prostate cancer. In certain embodiments,cancer-targeting peptides attached to a cytocidal, pro-apoptotic,anti-angiogenic or other therapeutic agent may be of use in methods totreat human cancer.

In other embodiments of the invention, the isolated peptide may beattached to a macromolecular complex. In preferred embodiments, themacromolecular complex is a virus, a bacteriophage, a bacterium, aliposome, a microparticle, a magnetic bead, a yeast cell, a mammaliancell, a cell or a microdevice. These are representative examples onlyand macromolecular complexes within the scope of the present inventionmay include virtually any complex that may be attached to a targetingpeptide and administered to a subject. In other preferred embodiments,the isolated peptide may be attached to a eukaryotic expression vector,more preferably a gene therapy vector.

Various embodiments concern novel targeted gene therapy vectors,comprising targeting peptides expressed on the surface of a gene therapyvector. In particular embodiments, the targeted gene therapy vector is achimeric phage-based vector containing elements from adeno-associatedvirus (AAV), the modified vector being referred to as anadeno-associated phage (AAP) vector.

In another embodiment, the targeting peptides may be attached to a solidsupport, preferably magnetic beads, Sepharose beads, agarose beads, anitrocellulose membrane, a nylon membrane, a column chromatographymatrix, a high performance liquid chromatography (HPLC) matrix or a fastperformance liquid chromatography (FPLC) matrix. Such immobilizedpeptides may be used, for example, for affinity purification of variouscomponents, such as receptor proteins or circulating antibodies thatbind to the peptides.

Additional embodiments of the present invention concern fusion proteinscomprising at least 3 contiguous amino acids of a sequence selected fromany of SEQ ID NO:5-35, SEQ ID NO:37, SEQ ID NO:39-67 and SEQ IDNO:83-129. In some embodiments, larger contiguous sequences, up to afull-length sequence selected from any of SEQ ID NO:5-35, SEQ ID NO:37,SEQ ID NO:39-67 and SEQ ID NO:83-129 may be used.

Certain other embodiments concern compositions comprising the claimedisolated peptides or fusion proteins in a pharmaceutically acceptablecarrier. Further embodiments concern kits comprising the claimedisolated peptides or fusion proteins in one or more containers.

Other embodiments concern methods of targeted delivery comprisingselecting a targeting peptide for a desired organ, tissue or cell type,such as prostate cancer, attaching said targeting peptide to a molecule,macromolecular complex or gene therapy vector, and providing saidpeptide attached to said molecule, complex or vector to a subject.Preferably, the targeting peptide is selected to include at least 3contiguous amino acids from any of selected from any of SEQ ID NO:5-35,SEQ ID NO:37 and SEQ ID NO:83-129. In other preferred embodiments, themolecule attached to the targeting peptide is a chemotherapeutic agent,an antigen or an imaging agent. In various embodiments, methods oftargeted delivery may utilize antibodies against particular peptidesequences, such as SEQ ID NO:39-67. Such antibodies may be attached to amolecule, macromolecular complex or gene therapy vector and administeredto a subject. The skilled artisan will realize that the targeting moietyis not limited to antibodies, but may comprise any molecule or complexthat binds to a receptor located in a target tissue, including but notlimited to antibodies, genetically engineered antibodies, antibodyfragments, single-chain antibodies, humanized antibodies, chimericantibodies, binding proteins and native ligands or homologs thereof. Inpreferred embodiments of the invention, the targeted receptor is GRP78or IL-11Rα.

In certain embodiments, the cancer targeting peptides and/or antibodiesdisclosed herein may be of use for the detection, diagnosis and/orprognosis of human cancer, such as prostate cancer. In preferredembodiments, the cancer targeting peptides may be used to differentiallydiagnose metastatic and non-metastatic prostate cancer.

Other embodiments of the present invention concern isolated nucleicacids of 300 nucleotides or less in size, encoding a targeting peptide.In preferred embodiments, the isolated nucleic acid is 250, 225, 200,175, 150, 125, 100, 75, 50, 40, 30, 20 or even 10 nucleotides or less insize. In other preferred embodiments, the isolated nucleic acid isincorporated into a eukaryotic or a prokaryotic expression vector. Ineven more preferred embodiments, the vector is a plasmid, a cosmid, ayeast artificial chromosome (YAC), a bacterial artificial chromosome(BAC), a virus or a bacteriophage. In other preferred embodiments, theisolated nucleic acid is operatively linked to a leader sequence thatlocalizes the expressed peptide to the extracellular surface of a hostcell.

Additional embodiments of the present invention concern methods oftreating a disease state, such as cancer, comprising selecting atargeting peptide and/or antibody against a selected peptide thattargets cells associated with the disease state, attaching one or moremolecules effective to treat the disease state to the peptide, andadministering the peptide to a subject with the disease state.Preferably, the peptide includes at least three contiguous amino acidsselected from any of selected from any of SEQ ID NO:5-35, SEQ ID NO:37,SEQ ID NO:39-67 and SEQ ID NO:83-129.

In certain embodiments, the methods concern Biopanning and RapidAnalysis of Selective Interactive Ligands (BRASIL), a novel method forphage display that results in decreased background of non-specific phagebinding, while retaining selective binding of phage to cell receptors.In preferred embodiments, targeting peptides are identified by exposinga subject to a phage display library, collecting samples of one or moreorgans, tissues or cell types, separating the samples into isolatedcells or small clumps of cells suspended in an aqueous phase, layeringthe aqueous phase over an organic phase, centrifuging the two phases sothat the cells are pelleted at the bottom of a centrifuge tube andcollecting phage from the pellet. In an even more preferred embodiment,the organic phase is dibutylphtalate.

In other embodiments, phage that bind to a target organ, tissue or celltype, for example to prostate cancer, may be pre-screened orpost-screened against a subject lacking that organ, tissue or cell type,such as a female subject. Phage that bind to a control subject areremoved from the library prior to screening in subjects possessing theorgan, tissue or cell type.

In preferred embodiments, targeting phage may be recovered from specificcell types or sub-types present in an organ or tissue after selection ofthe cell type by PALM (Positioning and Ablation with Laser Microbeams).PALM allows specific cell types to be selected from, for example, a thinsection of an organ or tissue. Phage may be recovered from the selectedsample.

In another embodiment, a phage display library displaying the antigenbinding portions of antibodies from a subject is prepared, the libraryis screened against one or more antigens and phage that bind to theantibodies are collected. In more preferred embodiments, the antigen isa targeting peptide.

In certain embodiments, the methods and compositions may be used toidentify one or more receptors for a targeting peptide. In alternativeembodiments, the compositions and methods may be used to identifynaturally occurring ligands for known or newly identified receptors. Inpreferred embodiments, the receptor may be selectively or specificallyexpressed in prostate cancer. In some embodiments, expression of thereceptor may be up regulated in prostate cancer compared to normalprostate, and/or in metastatic compared to non-metastatic prostatecancer. Methods of diagnosis and/or prognosis of cancer, such asprostate cancer, may comprise detection and/or quantification of suchdisease-state selective or specific receptors in tissue samples. In someembodiments, detection and/or quantification may take place in situwithin an intact subject, for example by attaching an imaging agent toan antibody or equivalent molecule that binds to the receptor.

In some embodiments, the methods may comprise contacting a targetingpeptide to an organ, tissue or cell containing a receptor of interest,allowing the peptide to bind to the receptor, and identifying thereceptor by its binding to the peptide. In preferred embodiments, thetargeting peptide contains at least three contiguous amino acidsselected from any of selected from any of SEQ ID NO:5-35, SEQ ID NO:37and SEQ ID NO:83-129. In other preferred embodiments, the targetingpeptide may comprise a portion of an antibody against the receptor. Inmore preferred embodiments, the antibody or antibody portion may bind toSEQ ID NO:39-67.

In alternative embodiments, the targeting peptide may contain a randomamino acid sequence. The skilled artisan will realize that thecontacting step can utilize intact organs, tissues or cells, or mayalternatively utilize homogenates or detergent extracts of the organs,tissues or cells. In certain embodiments, the cells to be contacted maybe genetically engineered to express a suspected receptor for thetargeting peptide. In a preferred embodiment, the targeting peptide ismodified with a reactive moiety that allows its covalent attachment tothe receptor. In a more preferred embodiment, the reactive moiety is aphotoreactive group that becomes covalently attached to the receptorwhen activated by light. In another preferred embodiment, the peptide isattached to a solid support and the receptor is purified by affinitychromatography. In other preferred embodiments, the solid supportcomprises magnetic beads, Sepharose beads, agarose beads, anitrocellulose membrane, a nylon membrane, a column chromatographymatrix, a high performance liquid chromatography (HPLC) matrix or a fastperformance liquid chromatography (FPLC) matrix.

In certain embodiments, the targeting peptide may inhibit the activityof a receptor upon binding to the receptor. The skilled artisan willrealize that receptor activity can be assayed by a variety of methodsknown in the art, including but not limited to catalytic activity andbinding activity. In other embodiments, binding of a targeting peptideto a receptor may inhibit a transport activity of the receptor.

In alternative embodiments, one or more ligands for a receptor ofinterest may be identified by the disclosed methods and compositions.One or more targeting peptides that mimic part or all of a naturallyoccurring ligand may be identified by phage display and biopanning invivo or in vitro. A naturally occurring ligand may be identified byhomology with a single targeting peptide that binds to the receptor, ora consensus motif of sequences that bind to the receptor. In otheralternative embodiments, an antibody may be prepared against one or moretargeting peptides that bind to a receptor of interest. Such antibodiesmay be used for identification or immunoaffinity purification of thenative ligand.

In certain embodiments, the targeting peptides of the present inventionare of use for the selective delivery of therapeutic agents, includingbut not limited to gene therapy vectors and fusion proteins, to specificorgans, tissues or cell types. The skilled artisan will realize that thescope of the claimed methods of use include any disease state that canbe treated by targeted delivery of a therapeutic agent to a desiredorgan, tissue or cell type. Although such disease states include thosewhere the diseased cells are confined to a specific organ, tissue orcell type, other disease states may be treated by an organ, tissue orcell type-targeting approach. In particular embodiments, the organ,tissue or cell type may comprise prostate cancer.

Certain embodiments concern methods of obtaining antibodies against anantigen. In preferred embodiments, the antigen comprises one or moretargeting peptides. The targeting peptides may be prepared andimmobilized on a solid support, serum-containing antibodies is added andantibodies that bind to the targeting peptides may be collected.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. IHC (immunohistochemistry) localization of IL-11Rα in benignprostate glands. (A) Normal glands from the peripheral zone showedpredominant nuclear staining of the basal and luminal cell layers(×200). (B) A similar pattern of staining was observed in normal glandsfrom the central zone (×200).

FIG. 2. IHC staining of IL-11Rα expression in primary androgen dependentprostate cancer of low, intermediate, and high Gleason scores (FIG.2A-C, respectively). (A) Gleason score 6 prostate adenocarcinoma showedhomogeneous 2+ staining (×200). (B) Prostate carcinoma (arrowheads)showing 1+ and 2+ heterogeneous staining. Note negative staining in theluminal cells of the contiguous benign glands (black arrows) (×100). (C)Strong 3+ positive staining in high-grade prostatic adenocarcinoma(×100). (D) Negative control including benign glands from the peripheralzone and a few neoplastic acini (×100).

FIG. 3. Immunodetection of IL-11Rα in advanced, androgen independent,prostate cancer. (A) Homogeneous 3+ expression of IL-11Rα in prostatecancer metastatic to the bone (×100). (B) A higher power view of a bonemetastasis showing 2+ and 3+ expression of the receptor in the tumorcells (×200). (C) Positive staining in the small vessels around thetumor nodules in the bone matrix (×200). (D) CD31 staining of theprevious area confirming endothelial cell reactivity (×200) (E)High-grade, androgen-independent primary tumor showing strong (3+) andhomogeneous expression of IL-11Rα (×100). (F) Negative control from thesame area as (B) (×100).

FIG. 4. Selection of peptide library on immunoglobulins from the serumof metastatic prostate cancer patients. Each successive round of panningdemonstrates an increase in selectivity as measured by the increase intotal number of transducing units for cancer patients relative to theserum of control volunteers. Three metastatic androgen-independents(patients A, B, and D) serum samples and one metastaticandrogen-dependent (patient C) serum sample were examined. Standarderror of the mean (S.E.M.) from triplicate plating is shown.

FIG. 5. Selection of peptide library on immunoglobulins from the serumof metastatic prostate cancer patients. A series of 100-fold dilutions(1:100-1:1200) was performed for each patient's serum to test specificbinding of cancer antibodies to immobilized GST-fusion proteins byELISA.

FIG. 6. Reactivity between the serum from prostate cancer patients orcontrol men and the selected peptide is stage-specific. Serum samplesderived from a large panel of prostate cancer patients (n=108) weredivided into four groups: organ-confined (n=17), locally advanced(n=31), and metastatic androgen-dependent (n=31), and metastaticandrogen-independent (n=29). Serum samples derived from 71 age-matchedblood-donor men served as a negative control group. Serial dilutionswere performed for each serum to determine optimal reactivity by ELISA.

FIG. 6A. Distribution of reactivity is shown as the ratio of GST-peptideto GST alone for the four prostate cancer groups and control. Positivereactivity was defined by a ratio of GST-peptide to GST alone equal toor greater than 2.

FIG. 6B. Distribution of reactivity is shown as a percentage of positivereactivity for each group. GST, glutathione S-transferase; A.D.,androgen-dependent; A.I., androgen-independent.

FIG. 6C. Correlation between overall survival and serological reactivityagainst the CNVSDKSC (SEQ ID NO:39) peptide. The same prostate cancerpatient population was used to generate the Kaplan-Meier survival curvesshown. Log-Rank tests were used to detect significant differences insurvival time between patients positively reacting versus non-reactingto the peptide. A significant correlation was observed between poorsurvival outcome and positive serum reactivity against the peptideCNVSDKSC (SEQ ID NO:39).

FIG. 7. Immunohistological analysis of tumors from a prostate cancerpatient. Immunostaining of sections from prostate cancer metastatic tothe bone marrow of the patient whose screening yielded CNVSDKSC (SEQ IDNO:39) and of normal prostate are shown. (A) Strong staining wasobserved on metastatic tumor with the autologous immunopurified IgGs.(B) Strong staining was also observed with a rabbit polyclonal antibodyraised against the synthetic form of the CNVSDKSC (SEQ ID NO:39) solublepeptide. (C) No staining was observed with the rabbit pre-immune serum.(D) No staining was observed with secondary antibody alone. (E) Arecombinant CNVSDKSC (SEQ ID NO:39) fusion protein inhibited stainingunder the same conditions used in FIG. 7B. (F) Weak staining wasobserved in normal prostate with the same rabbit polyclonal antibodyused in FIG. 7B. Scale bar is 50

FIG. 8. Cross-inhibition of patient serum antibodies by (A) GRP78 or (B)GST-CNVSDKSC (SEQ ID NO:39). Recombinant GRP78 or GST-CNVSDKSC (SEQ IDNO:39) were coated on microtiter well plates and various concentrationsof patient serum, anti-GRP78 antibody and anti-CNVSDKSC (SEQ ID NO:39)antibodies were added and analyzed by ELISA. Pre-incubation of thepatient serum antibodies with GRP78 or GST-CNVSDKSC (SEQ ID NO:39)inhibited the reaction. The data shows means±SD of triplicate wells.

FIG. 9. Reactivity against GRP78 is a serum marker of prostate cancer.(A) Microtiter wells were coated with recombinant GRP78 and triplicatesof serum samples were added at a 1:50 dilution. Serum samples from thesame prostate cancer population presented in FIG. 6 were examined. Forthis assay, male (n=155) and female (n=48) donors served as negativecontrols. Positive reactivity by ELISA was defined as an Absorbanceequal to or greater than 0.95 as determined by a statistical method“CART”. To test whether reactivity against GRP78 was restricted toprostate cancer, three additional non-prostate cancer tumor types areshown as controls: metastatic non-small cell (N.S.C.) lung cancer(n=31), metastatic breast cancer (n=32) and advanced ovarian cancer(32). Percentages of positive reactivity are shown. (B) CART test forcomparative survival of GRP78 reactive (lower line) versus non-reactive(upper line) individuals with prostate cancer.

FIG. 10. Expression pattern of GRP78 by immunohistochemistry.Immunostaining of normal prostate tissue and bone metastasis byanti-GRP78 antibody and anti-CNVSDKSC (SEQ ID NO:39) antibodies areshown. (A) Strong staining was observed in bone metastasis. (B) Weakstaining was observed in the normal prostate. (C) Recombinant GRP78 caninhibit staining. (D) Recombinant GST-CNVSDKSC (SEQ ID NO:39) alsoinhibits staining. The magnification-100×.

FIG. 11. Scheme of the construction of phage with a targeting domain anda mammalian reporter gene cassette. Replicative forms of thephage-derived RGD-4C and the fd-tet derived fMCS1 DNA were digested withSac II and Barn HI. Ligation of the fMCS1 fragment with the RGD-4Cplasmid fragment resulted in a chimeric RGD-4C-fMCS1 phage vector with amulticloning site containing a Pst I site. The Pst I-digested β-gal genecassette was cloned into the Pst I site of the chimeric vectorRGD-4C-fMCS1. The mammalian transgene cassette contains a CMV promoter,a β-galactosidase (β-gal) gene, and an SV40 polyadenylation signal (SV40polyA). The other targeted and control phage vectors presented in thisstudy were constructed by the same general strategy.

FIG. 12. Transgene expression in mammalian cells after transfection ofsingle-stranded phage DNA into 293 cells. β-gal expression was analyzedby an X-gal staining after 24 hours. (A) Positive control plasmidpCMVβ-gal. (B) Negative control plasmid without the reporter genecassette. (C) Single-stranded DNA extracted from phage with a forwardorientation of the transgene cassette. (D) Single-stranded DNA extractedfrom phage with a reverse orientation of the transgene cassette.

FIG. 13. Transduction of tumor cells by targeted phage is specific.Tumor cells were incubated with targeted phage. β-gal expression wasevaluated after 72 hours. An anti β-gal antibody (Sigma) was used forthe staining. (A, B) KS1767 cells with HWGF-β-gal phage, (C, D)MDA-MB-435 cells with RGD-4C-β-gal phage, (E, F) control insertlessphage (fd-tet-β-gal). The left side (A, C, E) shows only TexasRed-positive (β-gal infected) cells. The right side (B, D, E) shows thetotal number of cells in identical fields. Magnification: ×200.

FIG. 14. Quantitative analysis of cell transduction by targeted andcontrol phage. Phage were incubated with tumor cell lines as describedin the legend to FIG. 13. (A) An anti-β-gal antibody was used forstaining. Gene expression was detected by immunofluorescence and resultsare expressed in % of β-gal positive cells. In each case, standard errorof the mean (SEM) was calculated after counting 10 fields under themicroscope in three independent experiments. (B) Inhibition ofHWGF-β-gal phage transduction by the synthetic CTTHWGFTLC (SEQ ID NO:69)peptide. (C) Inhibition of RGD-4C-β-gal phage transduction by thesynthetic RGD-4C peptide. Unrelated control peptides did not inhibittransduction of the tumor cells by the targeted phage; non-specifictransduction levels were determined by using control insertless phage.Shown are mean±SEM obtained from duplicate wells.

FIG. 15. Specific transduction in vivo by tumor-targeting phage.Immunohistochemical analysis of β-gal expression after systemicadministration of targeted or control phage into tumor-bearing mice wasperformed. RGD-4C-β-gal (A, D, and G), HWGF-β-gal (B, E, and H), orcontrol phage (C, F, and I) were injected intravenously into micebearing KS1767-derived Kaposi's sarcoma xenografts. At seven dayspost-administration, tumors and control organs were removed, fixed in 4%paraformaldehyde, embedded in paraffin, and sectioned. An antibodyanti-β-gal (Sigma) was used for staining. Liver (D, E, and F) and brain(G, H, and I) are shown as control organs. Magnification: ×400. Arrowspoint to anti-β-gal immunoreactivity.

FIG. 16. Tumor-selective targeting by RGD-4C β-Gal phage, compared tocontrol insertless phage. The ability of different tissues to beinfected by the tumor targeting versus control phage was examined fortumor, kidney, lung, brain, liver and spleen tissues. Although acomparatively high level of RGD-4C phage were localized to kidney, thedifference between tumor-targeting and control phage distribution wasnot significant. Only tumor tissue showed a significant enhancement ofphage localization for the RGD-4C phage compared to control phage.

FIG. 17. Specific transduction in vivo by lung-targeting phage. Lung(targeted organ) and liver (control organ) were evaluated for β-galexpression after systemic administration of GFE-phage or control phageinto C57B1/6 immunocompetent mice. At 14 days post-administration lungsand livers were removed and processed as described in the text. β-galenzymatic activity in the tissue cell lysates was measured bychemiluminescence. Shown are mean±SEM (n=5 mice per group).

FIG. 18. Enhancement of transduction by genotoxic agents or genetictrans-complementation. Semi-confluent MDA-MB-435 cells were infectedwith 10⁵ TU of phage per cell for four hours. Next, the cells wereincubated for 36 hours followed by addition of genotoxic drugs(topotecan, 10 μM; cisplatin, 10 μM) or application of physical agentssuch as ultraviolet radiation (UV; 15 J/m²). A phage mixture ofRGD-4C-βgal forward and reverse clones (molar ratio=1; termed For/Rev)at the same number of phage TU of RGD-4C-βgal phage was also tested. At72 hours post-infection, the cells were analyzed for expression of areporter transgene. Shown are mean±SEM (n=3) normalized greenfluorescent protein (GFP) expression relative to controls.

FIG. 19. AAP vectors markedly improve gene transduction stability.Vectors were constructed by cloning a full-length 2.8 kb fragment ofpAAV-eGFP (Green Fluorescent Protein, Stratagene) from inverted terminalrepeat (ITR) to ITR into the blunted PstI site of the constructpresented in FIG. 11. An engineered chimeric vector composed of anRGD-4C targeted phage and AAV genetic cis-elements was incubated withcells and analyzed for GFP gene expression 72 hours after infection asindicated. Either synthetic RGD-4C peptide or control unrelated peptide(CKDRFERC, SEQ ID NO:41) was pre-incubated with cells to confirmspecificity of targeted gene transduction.

FIG. 20. GRP expression in cells infected with an AAV-GFP vector, in thepresence or absence of RGD-4C peptide or control peptide. For GFPdetection, cells in each experiment were analyzed by fluorescenceactivated cell sorting (FACS) and photographed under a fluorescencemicroscope.

FIG. 21. Time course of gene transduction. Cells were plated at 3×10⁵cells/well, infected with 10⁵ TU of phage per cell for 4 hours, andsorted based on GFP expression by FACS at seven days post-infection.GFP-positive cells were plated and GFP expression was monitored. RobustGFP expression is shown at days 0, 15, 30, and 45.

FIG. 22. AAP vectors promote AAV integration. Viral rescue experiments.GFP-expressing cells were detected after 48 hours. AAV particles can bedetected after adenoviral rescue in AAP-transduced 293 cells but not incontrol uninfected 293 cells incubated with culture medium.

FIG. 23. Validation of adipose homing peptides. Phage bearing targetingpeptides were injected into obese mice and their recovery from adiposetissue was compared to control fd-tet phage without targeting sequences.

FIG. 24. In vivo fat homing of the CKGGRAKDC (SEQ ID NO:81) motif ingenetically obese mice. (A) and (B) Anti-phage immunohistochemistry inparaffin sections of subcutaneous white fat from leptin-deficient miceintravenously injected 6 hr prior to tissue processing. (C) and (D)Peptide-FITC immunofluorescence in paraffin sections of subcutaneouswhite fat from leptin-deficient mice intravenously injected 6 hr priorto tissue processing. Mice were injected with (A) CKGGRAKDC (SEQ IDNO:81) phage, (B) control insertless phage, (C) CKGGRAKDC (SEQ ID NO:81)linked to FITC peptide, or (D) control CARAC (SEQ ID NO:71) linked toFITC peptide. Homing of the CKGGRAKDC (SEQ ID NO:81) peptide to fatblood vessels (arrows) and its uptake by fat endothelium are indicated.Bar: 10 μM.

FIG. 25. In vivo fat homing of the CKGGRAKDC (SEQ ID NO:81) motif inwild-type mice. (a), (C) and (E) Peptide-FITC immunofluorescence or (B),(D) and (F) lectin-rhodamine immunofluorescence in blood vessels of (A),(B), (E) and (F) subcutaneous white fat or (C) and (D) pancreas controlsdetected in paraffin-sectioned tissues from c57b1/6 mice intravenouslyco-injected 5 min prior to tissue processing. Mice were injected with(A), (B), (C) and (D) CKGGRAKDC (SEQ ID NO:81) linked to FITC peptideand lectin-rhodamine; or (E) and (F) control CARAC (SEQ ID NO:71) linkedto FITC peptide and lectin-rhodamine. (B), (D) and (F) Arrows showendothelium marked with lectin. (A) Arrows show homing of the CKGGRAKDC(SEQ ID NO:81) peptide to fat endothelium. Bar: 10

FIG. 26. Treatment of mouse obesity with fat vasculature-targetedapoptosis. Three cohorts (n=3) of (A) high-fat cafeteria diet-fed obesec57b1/6 mice; or (B) regular diet-fed old (˜1 year) c57b1/6 mice wereeach subcutaneously injected daily with equimolar amounts of theindicated peptides. Mouse body mass measurement was taken on days wheninjections were performed (injections were skipped on days for whichbody mass measurement is not shown). Error bars are SEM for themeasurements in three mice.

FIG. 27. Fat resorption induced by fat vasculature-targeted apoptosis.(A)

Representative high-fat cafeteria diet-fed obese c57b1/6 mice; (B) and(C) representative regular diet-fed old (˜1 year) c57b1/6 mice; or (D)epididymal fat from representative regular diet-fed old c57b1/6 micefrom the experiment described in FIG. 10. Whole mice (A), subcutaneousfat (B), peritoneal fat (C) and total epididymal fat (D) from thecorresponding indicated treatments were photographed 1 week (A) or 3weeks (B), (C) and (D) after the beginning of subcutaneous injections.The injected peptides were CKGGRAKDC (SEQ ID NO:81) linked to (KLAKLAK)₂(SEQ ID NO:1) (left column), CARAC (SEQ ID NO:71) linked to (KLAKLAK)₂(SEQ ID NO:1) (middle column), and CKGGRAKDC (SEQ ID NO:81)co-administered with (KLAKLAK)₂ (SEQ ID NO:1) (right column).

FIG. 28. Destruction of fat blood vessels as a result of targetedapoptosis. (A)

Tunnel immunohistochemistry, (B) secondary antibody only negative tunnelstaining control and (C) and (D) hematoxylin/eosin staining of white fatof mice. (A), (B) and (C) Mice were treated with CKGGRAKDC (SEQ IDNO:81) linked to (KLAKLAK)₂ (SEQ ID NO:1). (D) Mice were treated withCARAC (SEQ ID NO:71) linked to (KLAKLAK)₂ (SEQ ID NO:1). Apoptosis(arrows, (A)) and necrosis/lymphocyte infiltration (arrows, (C)) inresponse to CKGGRAKDC (SEQ ID NO:81) linked to (KLAKLAK)₂ (SEQ ID NO:1)treatment are indicated. Bar: 10 μm.

FIG. 29. Expression of prohibitin in human tissues. Prohibitinexpression was determined by immunohistochemistry of fixed humanparaffin-embedded thin tissue sections with rabbit polyclonal antibodiesagainst prohibitin. Arrows indicate prohibitin staining in: (A) normalhuman white fat tissue; (B) normal human breast tissue; (C) a low gradehuman lipoma; (D) a high grade human lipoma; (E) a myxoid liposarcoma;and (F) a dedifferentiated liposarcoma.

FIG. 30. Expression of prohibitin in human tissues. Prohibitinexpression was determined by immunohistochemistry of fixed humanparaffin-embedded thin tissue sections with rabbit polyclonal antibodiesagainst prohibitin. Arrows indicate prohibitin staining in normal humantissues of: (A) white fat; (B) skin; (C) prostate; (E) bone; and (F)muscle; and (F) skeletal muscle. A fat staining control is shown in (D).

FIG. 31. Model of prohibitin function in fat vasculature.

FIG. 32. AAP construction. The AAP vector was constructed as disclosedin Example 6.

FIG. 33. Distribution of IL-11Rα expression in primaryandrogen-dependent prostate carcinoma by immunohistochemical score,according to Gleason grade and pathological stage.

FIG. 34. Screening procedure for biopanning against ovarian cancerascites.

FIG. 35. Specificity of phage binding to ovarian cancer IgG vs. BSA orcontrol IgGs.

FIG. 36. Validation of ovarian cancer targeting by competition forbinding to IgGs isolated from ovarian cancer ascites to immobilized GSTfusion peptides versus the corresponding synthetic peptide (CVPELGHEC,SEQ ID NO:132).

FIG. 37. Reactivity between GST-CVPELGHEC (SEQ ID NO:132) fusion peptideand ascites from patients with different stages of ovarian cancer versusnon-ovarian cancer or non-malignant conditions. Positive reactivity isindicated as the ratio between binding to GST-fusion peptide compared toGST alone.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As used herein in the specification, “a” or “an” may mean one or more.As used herein in the claim(s), in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more of an item.

A “targeting peptide” is a peptide comprising a contiguous sequence ofamino acids, which is characterized by selective localization to anorgan, tissue or cell type. Selective localization may be determined,for example, by methods disclosed below, wherein the putative targetingpeptide sequence is incorporated into a protein that is displayed on theouter surface of a phage. Administration to a subject of a library ofsuch phage that have been genetically engineered to express a multitudeof such targeting peptides of different amino acid sequence is followedby collection of one or more organs, tissues or cell types from thesubject and identification of phage found in that organ, tissue or celltype. A phage expressing a targeting peptide sequence is considered tobe selectively localized to a tissue or organ if it exhibits greaterbinding in that tissue or organ compared to a control tissue or organ.Preferably, selective localization of a targeting peptide should resultin a two-fold or higher enrichment of the phage in the target organ,tissue or cell type, compared to a control organ, tissue or cell type.Selective localization resulting in at least a three-fold, four-fold,five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold orhigher enrichment in the target organ compared to a control organ,tissue or cell type is more preferred. Alternatively, a phage expressinga targeting peptide sequence that exhibits selective localizationpreferably shows an increased enrichment in the target organ compared toa control organ when phage recovered from the target organ arereinjected into a second host for another round of screening. Furtherenrichment may be exhibited following a third round of screening.Another alternative means to determine selective localization is thatphage expressing the putative target peptide preferably exhibit atwo-fold, more preferably a three-fold or higher enrichment in thetarget organ compared to control phage that express a non-specificpeptide or that have not been genetically engineered to express anyputative target peptides. Another means to determine selectivelocalization is that localization to the target organ of phageexpressing the target peptide is at least partially blocked by theco-administration of a synthetic peptide containing the target peptidesequence. “Targeting peptide” and “homing peptide” are used synonymouslyherein.

A “phage display library” means a collection of phage that have beengenetically engineered to express a set of putative targeting peptideson their outer surface. In preferred embodiments, DNA sequences encodingthe putative targeting peptides are inserted in frame into a geneencoding a phage capsule protein. In other preferred embodiments, theputative targeting peptide sequences are in part random mixtures of alltwenty amino acids and in part non-random. In certain preferredembodiments the putative targeting peptides of the phage display libraryexhibit one or more cysteine residues at fixed locations within thetargeting peptide sequence. Cysteines may be used, for example, tocreate a cyclic peptide.

A “macromolecular complex” refers to a collection of molecules that maybe random, ordered or partially ordered in their arrangement. The termencompasses biological organisms such as bacteriophage, viruses,bacteria, unicellular pathogenic organisms, multicellular pathogenicorganisms and prokaryotic or eukaryotic cells. The term also encompassesnon-living assemblages of molecules, such as liposomes, microcapsules,microparticles, magnetic beads and microdevices. The only requirement isthat the complex contains more than one molecule. The molecules may beidentical, or may differ from each other.

A “receptor” for a targeting peptide includes but is not limited to anymolecule or macromolecular complex that binds to a targeting peptide.Non-limiting examples of receptors include peptides, proteins,glycoproteins, lipoproteins, epitopes, lipids, carbohydrates,multi-molecular structures, a specific conformation of one or moremolecules and a morphoanatomic entity. In preferred embodiments, a“receptor” is a naturally occurring molecule or complex of moleculesthat is present on the lumenal surface of cells forming blood vesselswithin a target organ, tissue or cell type.

A “subject” refers generally to a mammal. In certain preferredembodiments, the subject is a mouse or rabbit. In even more preferredembodiments, the subject is a human.

Prostate Cancer Detection and Diagnosis

A particular problem in cancer detection and diagnosis occurs withprostate cancer. Carcinoma of the prostate (PCA) is the most frequentlydiagnosed cancer among men in the United States. Although relatively fewprostate tumors progress to clinical significance during the lifetime ofthe patient, those which are progressive in nature are likely to havemetastasized by the time of detection. Survival rates for individualswith metastatic prostate cancer are quite low. Between these extremesare patients with prostate tumors that will metastasize but have not yetdone so, for whom surgical prostate removal is curative. Determinationof which group a patient falls within is critical in determining optimaltreatment and patient survival.

Serum prostate specific antigen (PSA) is widely used as a biomarker todetect and monitor therapeutic response in prostate cancer patients(Badalament et al., 1996; O'Dowd et al., 1997). Although PSA has beenwidely used since 1988 as a clinical marker of prostate cancer (Partinand Oesterling, 1994), screening programs utilizing PSA alone or incombination with digital rectal examination (DRE) have not beensuccessful in improving the survival rate for men with prostate cancer(Partin and Oesterling, 1994). PSA is produced by normal and benign aswell as malignant prostatic tissue, resulting in a high false-positiverate for prostate cancer detection (Partin and Oesterling, 1994). Whilean effective indicator of prostate cancer when serum levels arerelatively high, PSA serum levels are more ambiguous indicators ofprostate cancer when only modestly elevated. The specificity of the PSAassay for prostate cancer detection at low serum PSA levels remains aproblem.

Other markers that have been used for prostate cancer detection includeprostatic acid phosphatase (PAP) (Brawn et al., 1996), prostate secretedprotein (PSP) (Huang et al., 1993), prostate specific membrane antigen(PSMA) (Murphy et al., 1995), human kallekrein 2 (HK2) (Piironen et al.,1996), prostate specific transglutaminase (pTGase) and interleukin 8(IL-8) (Veltri et al., 1999). None of these has yet been demonstrated toprovide a more sensitive and discriminating test for prostate cancerthan PSA.

In addition to these protein markers for prostate cancer, geneticchanges reported to be associated with prostate cancer, include allelicloss (Bova, et al., 1993); DNA hypermethylation (Isaacs et al., 1994);point mutations or deletions of the retinoblastoma (Rb), p53 and KAIlgenes (Isaacs et al., 1991); aneuploidy and aneusomy of chromosomesdetected by fluorescence in situ hybridization (FISH) (Macoska et al.,1994) and differential expression of HER2/neu oncogene receptor (An etal., 1998). None of these has been reported to exhibit sufficientsensitivity and specificity to be useful as general screening tools forasymptomatic prostate cancer.

In current clinical practice, the serum PSA assay and digital rectalexam (DRE) is used to indicate which patients should have a prostatebiopsy (Orozco et al., 1998). Histological examination of the biopsiedtissue is used to make the diagnosis of prostate cancer. It is estimatedthat over a half million prostate biopsies are performed annually in theUnited States (Orozco et al., 1998). A need exists for a serologicaltest that is sensitive enough to detect small and early stage prostatetumors, that also has sufficient specificity to exclude a greaterportion of patients with noncancerous conditions such as BPH.

There remain deficiencies in the prior art with respect to theidentification of markers linked with the progression of prostate cancerand the development of diagnostic methods to monitor diseaseprogression. The identification of novel, prostate selective or specificmarkers that are differentially expressed in metastatic and/ornon-metastatic prostate cancer, compared to non-malignant prostatetissue, would represent a major, unexpected advance for the diagnosis,prognosis and treatment of prostate cancer. As discussed below, oneapproach to identifying novel prostate cancer markers involves the phagedisplay technique. The skilled artisan will realize that althoughvarious embodiments of the invention are discussed in terms of prostatecancer, the disclosed methods and/or compositions may be of use toidentify markers (targeting peptides) for other types of cancer withinthe scope of the invention.

Phage Display

Recently, an in vivo selection system was developed using phage displaylibraries to identify organ, tissue or cell type-targeting peptides in amouse model system. Phage display libraries expressing transgenicpeptides on the surface of bacteriophage were initially developed to mapepitope binding sites of immunoglobulins (Smith, G P and Scott, J K,1985. Science, 228:1315-1317, Smith, G P and Scott, J K, 1993. Meth.Enzymol. 21:228-257). Such libraries can be generated by insertingrandom oligonucleotides into cDNAs encoding a phage surface protein,generating collections of phage particles displaying unique peptides inas many as 10⁹ permutations. (Pasqualini, R. and Ruoslahti, E. 1996,Nature, 380: 364-366; Arap et al, 1998a; Arap et al., 1998b, Curr. Opin.Oncol. 10:560-565).

Intravenous administration of phage display libraries to mice wasfollowed by the recovery of phage from individual organs (Pasqualini andRuoslahti, 1996). Phage were recovered that were capable of selectivehoming to the vascular beds of different mouse organs, tissues or celltypes, based on the specific targeting peptide sequences expressed onthe outer surface of the phage (Pasqualini and Ruoslahti, 1996). Avariety of organ and tumor-homing peptides have been identified by thismethod (Rajotte et al., 1998, J. Clin. Invest. 102:430-437; Rajotte etal, 1999, J. Biol. Chem. 274:11593-11598; Koivunen et al., 1999a, NatureBiotechnol. 17: 768-774; Burg M, et al., 1999a, Cancer Res.58:2869-2874; Pasqualini, 1999, Quart. J. Nucl. Med. 43:159-162). Eachof those targeting peptides bound to different receptors that wereselectively expressed on the vasculature of the mouse target tissue(Pasqualini, 1999; Pasqualini et al., 2000; Folkman J. NatureBiotechnol. 15:510, 1997; Folkman J. Nature Med 1:27-31, 1995). Inaddition to identifying individual targeting peptides selective for anorgan, tissue or cell type (Pasqualini and Ruoslahti, 1996; Arap et al,1998a; Koivunen et al., Methods Mol. Biol. 129: 3-17, 1999b), thissystem has been used to identify endothelial cell surface markers thatare expressed in mice in vivo (Rajotte and Ruoslahti, 1999).

Attachment of therapeutic agents to targeting peptides resulted in theselective delivery of the agent to a desired organ, tissue or cell typein the mouse model system. Targeted delivery of chemotherapeutic agentsand proapoptotic peptides to receptors located in tumor angiogenicvasculature resulted in an increase in therapeutic efficacy and adecrease in systemic toxicity in tumor bearing mouse models (Arap etal., 1998a, 1998b; Ellerby et al., Nature Med 9:1032-1038, 1999).

The methods described herein for identification of targeting peptidesinvolve the in vivo administration of phage display libraries. Variousmethods of phage display and methods for producing diverse populationsof peptides are well known in the art. For example, U.S. Pat. Nos.5,223,409; 5,622,699 and 6,068,829 disclose methods for preparing aphage library. The phage display technique involves geneticallymanipulating bacteriophage so that small peptides can be expressed ontheir surface (Smith and Scott, 1985, 1993). The past decade has seenconsiderable progress in the construction of phage-displayed peptidelibraries and in the development of screening methods in which thelibraries are used to isolate peptide ligands. For example, the use ofpeptide libraries has made it possible to characterize interacting sitesand receptor-ligand binding motifs within many proteins, such asantibodies involved in inflammatory reactions or integrins that mediatecellular adherence. This method has also been used to identify novelpeptide ligands that serve as leads to the development of peptidomimeticdrugs or imaging agents (Arap et al., 1998a). In addition to peptides,larger protein domains such as single-chain antibodies can also bedisplayed on the surface of phage particles (Arap et al., 1998a).

Targeting peptides selective for a given organ, tissue or cell type canbe isolated by “biopanning” (Pasqualini and Ruoslahti, 1996; Pasqualini,1999). In brief, a library of phage containing putative targetingpeptides is administered to an animal or human and samples of organs,tissues or cell types containing phage are collected. In preferredembodiments utilizing filamentous phage, the phage may be propagated invitro between rounds of biopanning in pilus-positive bacteria. Thebacteria are not lysed by the phage but rather secrete multiple copiesof phage that display a particular insert. Phage that bind to a targetmolecule can be eluted from the target organ, tissue or cell type andthen amplified by growing them in host bacteria. If desired, theamplified phage can be administered to a host and samples of organs,tissues or cell types again collected. Multiple rounds of biopanning canbe performed until a population of selective binders is obtained. Theamino acid sequence of the peptides is determined by sequencing the DNAcorresponding to the targeting peptide insert in the phage genome. Theidentified targeting peptide can then be produced as a synthetic peptideby standard protein chemistry techniques (Arap et al., 1998a, Smith andScott, 1985). This approach allows circulating targeting peptides to bedetected in an unbiased functional assay, without any preconceivednotions about the nature of their target. Once a candidate target isidentified as the receptor of a targeting peptide, it can be isolated,purified and cloned by using standard biochemical methods (Pasqualini,1999; Rajotte and Ruoslahti, 1999).

In certain embodiments, a subtraction protocol may be used withbiopanning to further reduce background phage binding. The purpose ofsubtraction is to remove phage from the library that bind to cells otherthan the cell of interest, or that bind to inactivated cells. Inalternative embodiments, the phage library may be prescreened against asubject who does not possess the targeted cell, tissue or organ. Forexample, prostate and/or prostate cancer binding peptides may beidentified after prescreening a library against female subjects. Aftersubtraction, the library may be screened against the cell, tissue ororgan of interest. In another alternative embodiment, an unstimulated,quiescent cell type, tissue or organ may be screened against the libraryand binding phage removed. The cell line, tissue or organ is thenactivated, for example by administration of a hormone, growth factor,cytokine or chemokine and the activated cell type, tissue or organscreened against the subtracted phage library. Other methods ofsubtraction protocols are known and may be used in the practice of thepresent invention, for example as disclosed in U.S. Pat. Nos. 5,840,841,5,705,610, 5,670,312 and 5,492,807.

Choice of Phage Display System.

Previous in vivo selection studies performed in mice preferentiallyemployed libraries of random peptides expressed as fusion proteins withthe gene III capsule protein in the fUSE5 vector (Pasqualini andRuoslahti, 1996). The number and diversity of individual clones presentin a given library is a significant factor for the success of in vivoselection. It is preferred to use primary libraries, which are lesslikely to have an over-representation of defective phage clones(Koivunen et al., 1999b). The preparation of a library should beoptimized to between 10⁸-10⁹ transducing units (T.U.)/ml. In certainembodiments, a bulk amplification strategy is applied between each roundof selection.

Phage libraries displaying linear, cyclic, or double cyclic peptides maybe used within the scope of the present invention. However, phagelibraries displaying 3 to 10 random residues in a cyclic insert(CX₃₋₁₀C) are preferred, since single cyclic peptides tend to have ahigher affinity for the target organ than linear peptides. Librariesdisplaying double-cyclic peptides (such as CX₃C X₃CX₃C; Rojotte et al.,1998) have been successfully used. However, the production of thecognate synthetic peptides, although possible, can be complex due to themultiple conformers with different disulfide bridge arrangements.

Identification of Homing Peptides and Receptors by In Vivo Phage Displayin Mice.

In vivo selection of peptides from phage-display peptide librariesadministered to mice has been used to identify targeting peptidesselective for normal mouse brain, kidney, lung, skin, pancreas, retina,intestine, uterus, prostate, and adrenal gland (Pasqualini andRuoslahti, 1996; Pasqualini, 1999; Rajotte et al., 1998). These resultsshow that the vascular endothelium of normal organs is sufficientlyheterogeneous to allow differential targeting with peptide probes(Pasqualini and Ruoslahti, 1996; Rajotte et al., 1998). A panel ofpeptide motifs that target the blood vessels of tumor xenografts in nudemice has been assembled (Arap et al., 1998a; reviewed in Pasqualini,1999). These motifs include the sequences RGD-4C, NGR, and GSL. TheRGD-4C peptide has previously been identified as selectively binding αvintegrins and has been reported to home to the vasculature of tumorxenografts in nude mice (Arap et al., 1998a, 1998b; Pasqualini et al.,Nature Biotechnol 15: 542-546, 1997).

The receptors for the tumor homing RGD4C targeting peptide has beenidentified as αv integrins (Pasqualini et al., 1997). The αv integrinsplay an important role in angiogenesis. The αvβ3 and αvβ5 integrins areabsent or expressed at low levels in normal endothelial cells and areinduced in angiogenic vasculature of tumors (Brooks P C, Clark R A,Cheresh D A. Science, 264: 569-571, 1994a; Hammes H P, Brownlee M,Jonczyk A, Sutter A, and Preissner K T. Nature Med. 2: 529-533, 1996.).Aminopeptidase N/CD13 has recently been identified as an angiogenicreceptor for the NGR motif (Burg, M. A., et al. Cancer Res. 59,2869-2874, 1999.). Aminopeptidase N/CD13 is strongly expressed not onlyin the angiogenic blood vessels of prostate cancer in TRAMP mice butalso in the normal epithelial prostate tissue.

Tumor-homing phage co-localize with their receptors in the angiogenicvasculature of tumors but not in non-angiogenic blood vessels in normaltissues (Arap et al., 1998b). Immunohistochemical evidence shows thatvascular targeting phage bind to human tumor blood vessels in tissuesections (Pasqualini et al., 2000) but not to normal blood vessels. Anegative control phage with no insert (fd phage) did not bind to normalor tumor tissue sections. The expression of the angiogenic receptors wasevaluated in cell lines, in non-proliferating blood vessels and inactivated blood vessels of tumors and other angiogenic tissues such ascorpus luteum. Flow cytometry and immunohistochemistry showed that thesereceptors are expressed in a number of tumor cells and in activatedHUVECs (data not shown). The angiogenic receptors were not detected inthe vasculature of normal organs of mouse or human tissues.

The distribution of these receptors was analyzed by immunohistochemistryin tumor cells, tumor vasculature, and normal vasculature. Alpha vintegrins, CD13, aminopeptidase A, NG2, and MMP-2/MMP-9—the knownreceptors in tumor blood vessels—are specifically expressed inangiogenic endothelial cells and pericytes of both human and murineorigin. Angiogenic neovasculature expresses markers that are eitherexpressed at very low levels or not at all in non-proliferatingendothelial cells (not shown).

The markers of angiogenic endothelium include receptors for vasculargrowth factors, such as specific subtypes of VEGF and basic FGFreceptors, and αv integrins, among many others (Mustonen T and AlitaloK. J. Cell Biol. 129:895-898, 1995.). Thus far, identification andisolation of novel molecules characteristic of angiogenic vasculaturehas been slow, mainly because endothelial cells undergo dramaticphenotypic changes when grown in culture (Watson et al., Science,268:447-448, 1995).

Many of these tumor vascular markers are proteases and some of themarkers also serve as viral receptors. Alpha v integrins are receptorsfor adenoviruses (Wickham et al., Cancer Immunol. Immunother.45:149-151, 1997c) and CD13 is a receptor for coronaviruses (Look et al.N. J. Clin. Invest. 83:1299-1307, 1989.). MMP-2 and MMP-9 are receptorsfor echoviruses (Koivunen et al., 1999a). Aminopeptidase A also appearsto be a viral receptor. Bacteriophage may use the same cellularreceptors as eukaryotic viruses. These findings suggest that receptorsisolated by in vivo phage display will have cell internalizationcapability, a key feature for utilizing the identified peptide motifs astargeted gene therapy carriers.

Targeted Delivery

Peptides that home to tumor vasculature have been coupled to cytotoxicdrugs or proapoptotic peptides to yield compounds that were moreeffective and less toxic than the parental compounds in experimentalmodels of mice bearing tumor xenografts (Arap et al., 1998a; Ellerby etal, 1999). The insertion of the RGD-4C peptide into a surface protein ofan adenovirus has produced an adenoviral vector that may be of use fortumor targeted gene therapy (Arap et al., 1998b). A need exists forimproved gene therapy vectors capable of targeted delivery in humansubjects, particularly for improved vectors that exhibit prolongedexpression of therapeutic genes in the transfected cells.

BRASIL

In preferred embodiments, separation of phage bound to the cells of atarget organ, tissue or cell type from unbound phage is achieved usingthe BRASIL technique (PCT Patent Application PCT/US01/28124 entitled,“Biopanning and Rapid Analysis of Selective Interactive Ligands(BRASIL)” by Arap et al., filed Sep. 7, 2001, incorporated herein byreference in its entirety). In BRASIL (Biopanning and Rapid Analysis ofSoluble Interactive Ligands), an organ, tissue or cell type is gentlyseparated into cells or small clumps of cells that are suspended in anaqueous phase. The aqueous phase is layered over an organic phase ofappropriate density and centrifuged. Cells attached to bound phage arepelleted at the bottom of the centrifuge tube, while unbound phageremain in the aqueous phase. This allows a more efficient separation ofbound from unbound phage, while maintaining the binding interactionbetween phage and cell. BRASIL may be performed in an in vivo protocol,in which organs, tissues or cell types are exposed to a phage displaylibrary by intravenous administration, or by an ex vivo protocol, wherethe cells are exposed to the phage library in the aqueous phase beforecentrifugation. A non-limiting exemplary application of the BRASILtechnique is disclosed in the Examples below.

Preparation of Large Scale Primary Libraries

In certain embodiments, primary phage libraries are amplified beforeinjection into a human subject. A phage library is prepared by ligatingtargeting peptide-encoding sequences into a phage vector, such as fUSE5.The vector is transformed into pilus negative host E. coli such asstrain MC1061. The bacteria are grown overnight and then aliquots arefrozen to provide stock for library production. Use of pilus negativebacteria αvoids the bias in libraries that arises from differentialinfection of pilus positive bacteria by different targeting peptidesequences.

To freeze, bacteria are pelleted from two thirds of a primary libraryculture (5 liters) at 4000×g for 10 min. Bacteria are resuspended andwashed twice with 500 ml of 10% glycerol in water, then frozen in anethanol/dry ice bath and stored at −80° C.

For amplification, 1.5 ml of frozen bacteria are inoculated into 5liters of LB medium with 20 μg/ml tetracycline and grown overnight.Thirty minutes after inoculation, a serial dilution is plated on LB/tetplates to verify the viability of the culture. If the number of viablebacteria is less than 5-10 times the number of individual clones in thelibrary (1−2×10⁸) the culture is discarded.

After growing the bacterial culture overnight, phage are precipitated.About ¼ to ⅓ of the bacterial culture is kept growing overnight in 5liters of fresh medium and the cycle is repeated up to 5 times. Phageare pooled from all cycles and used for injection into human subjects.

Human Subjects

The methods used for phage display biopanning in the mouse model systemrequire substantial improvements for use with humans. Techniques forbiopanning in human subjects are disclosed in PCT Patent ApplicationPCT/US01/28044, filed Sep. 7, 2001, the entire text of which isincorporated herein by reference. In general, humans suitable for usewith phage display are either brain dead or terminal wean patients. Theamount of phage library (preferably primary library) required foradministration must be significantly increased, preferably to 10¹⁴ TU orhigher, preferably administered intravenously in approximately 200 ml ofRinger lactate solution over about a 10 minute period.

The amount of phage required for use in humans has required substantialimprovement of the mouse protocol, increasing the amount of phageavailable for injection by five orders of magnitude. To produce suchlarge phage libraries, the transformed bacterial pellets recovered fromup to 500 to 1000 transformations are amplified up to 10 times in thebacterial host, recovering the phage from each round of amplificationand adding LB Tet medium to the bacterial pellet for collection ofadditional phage. The phage inserts remain stable under these conditionsand phage may be pooled to form the large phage display library requiredfor humans.

Samples of various organs and tissues are collected startingapproximately 15 minutes after injection of the phage library. Samplesare processed as described below and phage collected from each organ,tissue or cell type of interest for DNA sequencing to determine theamino acid sequences of targeting peptides.

With humans, the opportunities for enrichment by multiple rounds ofbiopanning are severely restricted, compared to the mouse model system.A substantial improvement in the biopanning technique involves polyorgantargeting.

Polyorgan Targeting

In the standard protocol for phage display biopanning, phage from asingle organ are collected, amplified and injected into a new host,where tissue from the same organ is collected for phage rescue and a newround of biopanning. This protocol is feasible in animal subjects.However, the limited availability and expense of processing samples fromhumans requires an improvement in the protocol.

It is possible to pool phage collected from multiple organs after afirst round of biopanning and inject the pooled sample into a newsubject, where each of the multiple organs may be collected again forphage rescue. The polyorgan targeting protocol may be repeated for asmany rounds of biopanning as desired. In this manner, it is possible tosignificantly reduce the number of subjects required for isolation oftargeting peptides for multiple organs, while still achievingsubstantial enrichment of the organ-homing phage.

In preferred embodiments, phage are recovered from human organs, tissuesor cell types after injection of a phage display library into a humansubject. In certain embodiments, phage may be recovered by exposing asample of the organ, tissue or cell type to a pilus positive bacterium,such as E. coli K91. In alternative embodiments, phage may be recoveredby amplifying the phage inserts, ligating the inserts to phage DNA andproducing new phage from the ligated DNA.

Proteins and Peptides

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide. As used herein,a protein or peptide generally refers, but is not limited to, a proteinof greater than about 200 amino acids up to a full length sequencetranslated from a gene; a polypeptide of about 100 to 200 amino acids;and/or a peptide of from about 3 to about 100 amino acids. Forconvenience, the terms “protein,” “polypeptide” and “peptide are usedinterchangeably herein.

In certain embodiments the size of at least one protein or peptide maycomprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about110, about 120, about 130, about 140, about 150, about 160, about 170,about 180, about 190, about 200, about 210, about 220, about 230, about240, about 250, about 275, about 300, about 325, about 350, about 375,about 400, about 425, about 450, about 475, about 500, about 525, about550, about 575, about 600, about 625, about 650, about 675, about 700,about 725, about 750, about 775, about 800, about 825, about 850, about875, about 900, about 925, about 950, about 975, about 1000, about 1100,about 1200, about 1300, about 1400, about 1500, about 1750, about 2000,about 2250, about 2500 or greater amino acid residues.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid, including but not limited to those shown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids bbr. Amino Acid Abbr. AminoAcid ad 2-Aminoadipic acid EtAsn N-Ethylasparagine aad 3-Aminoadipicacid Hyl Hydroxylysine ala β-alanine, AHyl allo-Hydroxylysineβ-Amino-propionic acid bu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline Abu4-Aminobutyric acid 4Hyp 4-Hydroxyproline piperidinic acid, cp6-Aminocaproic acid Ide Isodesmosine he 2-Aminoheptanoic acid AIleallo-Isoleucine ib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine aib 3-Aminoisobutyric acid MeIle N-Methylisoleucine pm2-Aminopimelic acid MeLys 6-N-Methyllysine bu 2,4-Diaminobutyric acidMeVal N-Methylvaline es Desmosine Nva Norvaline pm 2,2′-Diaminopimelicacid Nle Norleucine pr 2,3-Diaminopropionic acid Orn Ornithine tGlyN-Ethylglycine

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases(http://www.ncbi.nlm.nih.gov/). The coding regions for known genes maybe amplified and/or expressed using the techniques disclosed herein oras would be know to those of ordinary skill in the art. Alternatively,various commercial preparations of proteins, polypeptides and peptidesare known to those of skill in the art.

Peptide Mimetics

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., “Peptide Turn Mimetics” inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993), incorporated herein by reference. The underlying rationalebehind the use of peptide mimetics is that the peptide backbone ofproteins exists chiefly to orient amino acid side chains in such a wayas to facilitate molecular interactions, such as those of antibody andantigen. A peptide mimetic is expected to permit molecular interactionssimilar to the natural molecule. These principles may be used toengineer second generation molecules having many of the naturalproperties of the targeting peptides disclosed herein, but with alteredand even improved characteristics.

Fusion Proteins

Other embodiments of the present invention concern fusion proteins.These molecules generally have all or a substantial portion of atargeting peptide, linked at the N- or C-terminus, to all or a portionof a second polypeptide or protein. For example, fusions may employleader sequences from other species to permit the recombinant expressionof a protein in a heterologous host. Another useful fusion includes theaddition of an immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions. In preferred embodiments, the fusion proteins ofthe instant invention comprise a targeting peptide linked to atherapeutic protein or peptide. Examples of proteins or peptides thatmay be incorporated into a fusion protein include cytostatic proteins,cytocidal proteins, pro-apoptosis agents, anti-angiogenic agents,hormones, cytokines, growth factors, peptide drugs, antibodies, Fabfragments antibodies, antigens, receptor proteins, enzymes, lectins, MHCproteins, cell adhesion proteins and binding proteins. These examplesare not meant to be limiting and it is contemplated that within thescope of the present invention virtually and protein or peptide could beincorporated into a fusion protein comprising a targeting peptide.Methods of generating fusion proteins are well known to those of skillin the art. Such proteins can be produced, for example, by chemicalattachment using bifunctional cross-linking reagents, by de novosynthesis of the complete fusion protein, or by attachment of a DNAsequence encoding the targeting peptide to a DNA sequence encoding thesecond peptide or protein, followed by expression of the intact fusionprotein.

Protein Purification

In certain embodiments a protein or peptide may be isolated or purified.Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, gel exclusionchromatography, polyacrylamide gel electrophoresis, affinitychromatography, immunoaffinity chromatography, reverse phasechromatography and isoelectric focusing. An example of receptor proteinpurification by affinity chromatography is disclosed in U.S. Pat. No.5,206,347, the entire text of which is incorporated herein by reference.A particularly efficient method of purifying peptides is fastperformance liquid chromatography (FPLC) or even high performance liquidchromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by: centrifugation; chromatography stepssuch as ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

Affinity chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculeto which it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that itself does not adsorb molecules toany significant extent and that has a broad range of chemical, physicaland thermal stability. The ligand should be coupled in such a way as tonot affect its binding properties. The ligand should also providerelatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand.

Synthetic Peptides

Because of their relatively small size, the targeting peptides of theinvention can be synthesized in solution or on a solid support inaccordance with conventional techniques. Various automatic synthesizersare commercially available and can be used in accordance with knownprotocols. See, for example, Stewart and Young, Solid Phase PeptideSynthesis, 2d ed. Pierce Chemical Co., 1984; Tam et al., J. Am. Chem.Soc., 105:6442, 1983; Merrifield, Science, 232: 341-347, 1986; andBarany and Merrifield, The Peptides, Gross and Meienhofer, eds.,Academic Press, New York, pp. 1-284, 1979, each incorporated herein byreference. Short peptide sequences, usually from about 6 up to about 35to 50 amino acids, can be readily synthesized by such methods.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of the invention is insertedinto an expression vector, transformed or transfected into anappropriate host cell, and cultivated under conditions suitable forexpression.

Antibodies

In certain embodiments, it may be desirable to make antibodies againstthe identified targeting peptides or their receptors. The appropriatetargeting peptide or receptor, or portions thereof, may be coupled,bonded, bound, conjugated, or chemically-linked to one or more agentsvia linkers, polylinkers, or derivatized amino acids. This may beperformed such that a bispecific or multivalent composition or vaccineis produced. It is further envisioned that the methods used in thepreparation of these compositions are familiar to those of skill in theart and should be suitable for administration to humans, i.e.,pharmaceutically acceptable. Preferred agents are the carriers arekeyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA).

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. Techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; incorporated herein by reference).

In various embodiments of the invention, circulating antibodies from oneor more individuals with a disease state may be obtained and screenedagainst phage display libraries. Targeting peptides that bind to thecirculating antibodies may act as mimeotopes of a native antigen, suchas a receptor protein located on an endothelial cell surface of a targettissue. For example, circulating antibodies in an individual withprostate cancer may bind to antigens specifically or selectivelylocalized in prostate tumors. As discussed in more detail below,targeting peptides against such antibodies may be identified by phagedisplay. Such targeting peptides may be used to identify the nativeantigen recognized by the antibodies, for example by using knowntechniques such as immunoaffinity purification, Western blotting,electrophoresis followed by band excision and protein/peptide sequencingand/or computerized homology searches. The skilled artisan will realizethat antibodies against disease specific or selective antigens may be ofuse for various applications, such as detection, diagnosis and/orprognosis of a disease state, imaging of diseased tissues and/ortargeted delivery of therapeutic agents.

Imaging Agents and Radioisotopes

In certain embodiments, the claimed peptides or proteins of the presentinvention may be attached to imaging agents of use for imaging anddiagnosis of various diseased organs, tissues or cell types. Forexample, a prostate cancer selective targeting peptide may be attachedto an imaging agent, provided to a subject and the precise boundaries ofthe cancer tissue may be determined by standard imaging techniques, suchas CT scanning, MRI, PET scanning, etc. Alternatively, the presence orabsence and location in the body of metastatic prostate cancer may bedetermined by imaging using one or more targeting peptides that areselective for metastatic prostate cancer. Targeting peptides that bindto normal as well as cancerous prostate tissues may still be of use, assuch peptides would not be expected to be selectively localized anywherebesides the prostate in disease-free individuals. Naturally, thedistribution of a prostate or prostate cancer selective targetingpeptide may be compared to the distribution of one or more non-selectivepeptides to provide even greater discrimination for detection and/orlocalization of diseased tissues.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to proteins or peptides (see, e.g., U.S. Pat. Nos.5,021,236 and 4,472,509, both incorporated herein by reference). Certainattachment methods involve the use of a metal chelate complex employing,for example, an organic chelating agent such a DTPA attached to theprotein or peptide (U.S. Pat. No. 4,472,509). Proteins or peptides alsomay be reacted with an enzyme in the presence of a coupling agent suchas glutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate.

Non-limiting examples of paramagnetic ions of potential use as imagingagents include chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and erbium (III), with gadolinium beingparticularly preferred. Ions useful in other contexts, such as X-rayimaging, include but are not limited to lanthanum (III), gold (III),lead (II), and especially bismuth (III).

Radioisotopes of potential use as imaging or therapeutic agents includeastatine²¹¹, ¹⁴-carbon, ⁵¹chromium, ³⁶-chlorine, ⁵⁷cobalt, ⁵⁸cobalt,copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen, iodine¹²³, iodine¹²⁵, iodine¹³¹,indium¹¹¹, ⁵⁹iron, ³² phosphorus, rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium,³⁵sulphur, technicium^(99m) and yttrium⁹⁰. ¹²⁵I is often being preferredfor use in certain embodiments, and technicium^(99m) and indium¹¹¹ arealso often preferred due to their low energy and suitability for longrange detection.

Radioactively labeled proteins or peptides of the present invention maybe produced according to well-known methods in the art. For instance,they can be iodinated by contact with sodium or potassium iodide and achemical oxidizing agent such as sodium hypochlorite, or an enzymaticoxidizing agent, such as lactoperoxidase. Proteins or peptides accordingto the invention may be labeled with technetium-^(99m) by ligandexchange process, for example, by reducing pertechnate with stannoussolution, chelating the reduced technetium onto a Sephadex column andapplying the peptide to this column or by direct labeling techniques,e.g., by incubating pertechnate, a reducing agent such as SNCl₂, abuffer solution such as sodium-potassium phthalate solution, and thepeptide. Intermediary functional groups that are often used to bindradioisotopes that exist as metallic ions to peptides arediethylenetriaminepenta-acetic acid (DTPA) and ethylenediaminetetra-acetic acid (EDTA). Also contemplated for use arefluorescent labels, including rhodamine, fluorescein isothiocyanate andrenographin.

In certain embodiments, the claimed proteins or peptides may be linkedto a secondary binding ligand or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of suitable enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase. Preferredsecondary binding ligands are biotin and avidin or streptavidincompounds. The use of such labels is well known to those of skill in theart in light and is described, for example, in U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241;each incorporated herein by reference.

Cross-Linkers

The targeting peptides, ligands, receptor proteins and other moleculesof interest may be attached to surfaces or to therapeutic agents andother molecules using a variety of known cross-linking agents. Methodsfor covalent or non-covalen attachment of proteins or peptides, are wellknown in the art. Such methods may include, but are not limited to, useof chemical cross-linkers, photoactivated cross-linkers and/orbifunctional cross-linking reagents. Exemplary methods for cross-linkingmolecules are disclosed in U.S. Pat. No. 5,603,872 and U.S. Pat. No.5,401,511, incorporated herein by reference. Non-limiting examples ofcross-linking reagents of potential use include glutaraldehyde,bifunctional oxirane, ethylene glycol diglycidyl ether, carbodiimidessuch as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ordicyclohexylcarbodiimide, bisimidates, dinitrobenzene,N-hydroxysuccinimide ester of suberic acid, disuccinimidyl tartarate,dimethyl-3,3′-dithio-bispropionimidate, azidoglyoxal,N-succinimidyl-3-(2-pyridyldithio)propionate and4-(bromoadminoethyl)-2-nitrophenylazide.

Homobifunctional reagents that carry two identical functional groups arehighly efficient in inducing cross-linking. Heterobifunctional reagentscontain two different functional groups. By taking advantage of thedifferential reactivities of the two different functional groups,cross-linking can be controlled both selectively and sequentially. Thebifunctional cross-linking reagents can be divided according to thespecificity of their functional groups, e.g., amino, sulfhydryl,guanidino, indole, carboxyl specific groups. Of these, reagents directedto free amino groups have become especially popular because of theircommercial availability, ease of synthesis and the mild reactionconditions under which they can be applied.

In certain embodiments, it may be appropriate to link one or moretargeting peptides to a liposome or other membrane-bounded particle. Forexample, targeting peptides cross-linked to liposomes, microspheres orother such devices may be used to deliver larger volumes of atherapeutic agent to a target organ, tissue or cell type. Variousligands can be covalently bound to liposomal surfaces through thecross-linking of amine residues. Liposomes containingphosphatidylethanolamine (PE) may be prepared by established procedures.The inclusion of PE provides an active functional amine residue on theliposomal surface.

In another non-limiting example, heterobifunctional cross-linkingreagents and methods of use are disclosed in U.S. Pat. No. 5,889,155,incorporated herein by reference. The cross-linking reagents combine anucleophilic hydrazide residue with an electrophilic maleimide residue,allowing coupling in one example, of aldehydes to free thiols. Thecross-linking reagent can be modified to cross-link various functionalgroups.

Other techniques of general use for proteins or peptides that are knownin the art have not been specifically disclosed herein, but may be usedin the practice of the claimed subject matter.

Nucleic Acids

In certain embodiments, nucleic acids may encode a targeting peptide, areceptor protein, a fusion protein or other protein or peptide. Thenucleic acid may be derived from genomic DNA, complementary DNA (cDNA)or synthetic DNA. Where incorporation into an expression vector isdesired, the nucleic acid may also comprise a natural intron or anintron derived from another gene. Such engineered molecules are sometimereferred to as “mini-genes.” In various embodiments of the invention,targeting peptides may be incorporated into gene therapy vectors vianucleic acids.

A “nucleic acid” as used herein includes single-stranded anddouble-stranded molecules, as well as DNA, RNA, chemically modifiednucleic acids and nucleic acid analogs. It is contemplated that anucleic acid within the scope of the present invention may be of almostany size, determined in part by the length of the encoded protein orpeptide.

It is contemplated that targeting peptides, fusion proteins andreceptors may be encoded by any nucleic acid sequence that encodes theappropriate amino acid sequence. The design and production of nucleicacids encoding a desired amino acid sequence is well known to those ofskill in the art, using standardized codon tables (see Table 2 below).In preferred embodiments, the codons selected for encoding each aminoacid may be modified to optimize expression of the nucleic acid in thehost cell of interest. Codon preferences for various species of hostcell are well known in the art.

TABLE 2 Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In addition to nucleic acids encoding the desired peptide or protein,the present invention encompasses complementary nucleic acids thathybridize under high stringency conditions with such coding nucleic acidsequences. High stringency conditions for nucleic acid hybridization arewell known in the art. For example, conditions may comprise low saltand/or high temperature conditions, such as provided by about 0.02 M toabout 0.15 M NaCl at temperatures of about 50° C. to about 70° C. It isunderstood that the temperature and ionic strength of a desiredstringency are determined in part by the length of the particularnucleic acid(s), the length and nucleotide content of the targetsequence(s), the charge composition of the nucleic acid(s), and to thepresence or concentration of formamide, tetramethylammonium chloride orother solvent(s) in a hybridization mixture.

Nucleic acids for use in the disclosed methods and compositions may beproduced by any method known in the art, such as chemical synthesis(e.g. Applied Biosystems Model 3900, Foster City, Calif.), purchase fromcommercial sources (e.g. Midland Certified Reagents, Midland, Tex.)and/or standard gene cloning methods. A number of nucleic acid vectors,such as expression vectors and/or gene therapy vectors, may becommercially obtained (e.g., American Type Culture Collection,Rockville, Md.; Promega Corp., Madison, Wis.; Stratagene, La Jolla,Calif.).

Vectors for Cloning, Gene Transfer and Expression

In certain embodiments expression vectors are employed to express thetargeting peptide or fusion protein, which can then be purified andused. In other embodiments, the expression vectors are used in genetherapy. Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare known.

Regulatory Elements

The terms “expression construct” or “expression vector” are meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid codingsequence is capable of being transcribed. In preferred embodiments, thenucleic acid encoding a gene product is under transcriptional control ofa promoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrase“under transcriptional control” means that the promoter is in thecorrect location and orientation in relation to the nucleic acid tocontrol RNA polymerase initiation and expression of the gene.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent and under the controlof a promoter that transcriptionally active in human cells. Generallyspeaking, such a promoter might include either a human or viralpromoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rouse sarcoma virus longterminal repeat, rat insulin promoter, and glyceraldehyde-3-phosphatedehydrogenase promoter can be used to obtain high-level expression ofthe coding sequence of interest. The use of other viral or mammaliancellular or bacterial phage promoters that are known in the art toachieve expression of a coding sequence of interest is contemplated aswell, provided that the levels of expression are sufficient for a givenpurpose.

Where a cDNA insert is employed, one will typically include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed, such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression construct is a terminator. These elements can serve toenhance message levels and to minimize read through from the constructinto other sequences.

Selectable Markers

In certain embodiments of the invention, the cells containing nucleicacid constructs of the present invention may be identified in vitro orin vivo by including a marker in the expression construct. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression construct. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants. For example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Immunologic markers also can be employed. Theselectable marker employed is not believed to be important, so long asit is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art.

Delivery of Expression Vectors

There are a number of ways in which expression vectors may introducedinto cells. In certain embodiments of the invention, the expressionconstruct comprises a virus or engineered construct derived from a viralgenome. The ability of certain viruses to enter cells viareceptor-mediated endocytosis, to integrate into host cell genome, andexpress viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign genes into mammalian cells(Ridgeway, 1988; Nicolas and Rubinstein, In: Vectors: A survey ofmolecular cloning vectors and their uses, Rodriguez and Denhardt, eds.,Stoneham: Butterworth, pp. 494-513, 1988.; Baichwal and Sugden,Baichwal, In: Gene Transfer, Kucherlapati R, ed., New York, PlenumPress, pp. 117-148, 1986. 1986; Temin, In: Gene Transfer, Kucherlapati,R. ed., New York, Plenum Press, pp. 149-188, 1986). Preferred genetherapy vectors are generally viral vectors.

In using viral delivery systems, one will desire to purify the virionsufficiently to render it essentially free of undesirable contaminants,such as defective interfering viral particles or endotoxins and otherpyrogens such that it will not cause any untoward reactions in the cell,animal or individual receiving the vector construct. A preferred meansof purifying the vector involves the use of buoyant density gradients,such as cesium chloride gradient centrifugation.

DNA viruses used as gene vectors include the papovaviruses (e.g., simianvirus 40, bovine papilloma virus, and polyoma) (Ridgeway, pp 467-492,1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988;Baichwal and Sugden, 1986).

An exemplary method for in vivo delivery involves the use of anadenovirus expression vector. Although adenovirus vectors have a lowcapacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include, butis not limited to, constructs containing adenovirus sequences sufficientto (a) support packaging of the construct and (b) to express anantisense or a sense polynucleotide that has been cloned therein.

Generation and propagation of adenovirus vectors that are replicationdeficient depend on a helper cell line, such as the 293 cell line, whichwas transformed from human embryonic kidney cells by Ad5 DNA fragmentsand constitutively expresses E1 proteins (Graham et al., J. Gen. Virol.,36:59-72, 1977.). Since the E3 region is dispensable from the adenovirusgenome (Jones and Shenk, Cell, 13:181-188, 1978), adenovirus vectors,with the help of 293 cells, carry foreign DNA in either the E1, the E3,or both regions (Graham and Prevec, In: Methods in Molecular Biology:Gene Transfer and Expression Protocol, E. J. Murray, ed., Humana Press,Clifton, N.J., 7:109-128, 1991.).

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells or other humanembryonic mesenchymal or epithelial cells. Alternatively, the helpercells may be derived from the cells of other mammalian species that arepermissive for human adenovirus. Such cells include, e.g., Vero cells orother monkey embryonic mesenchymal or epithelial cells. Racher et al.,(Biotechnol. Tech. 9:169-174, 1995) disclosed methods for culturing 293cells and propagating adenovirus.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., Gene, 101:195-202, 1991; Gomez-Foix et al., J. Biol. Chem.,267:25129-25134, 1992) and vaccine development (Grunhaus and Horwitz,1992; Graham and Prevec, 1991). Animal studies have suggested thatrecombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, In: Human Gene Transfer, O.Cohen-Haguenauer et al, eds. John Libbey Eurotext, France, pp. 51-61,1991; Stratford-Perricaudet et al., Hum. Gene Ther. 1:241-256, 1990;Rich et al., Hum. Gene. Ther. 4:461-476, 1993). Studies in administeringrecombinant adenovirus to different tissues include trachea instillation(Rosenfeld et al., Science, 252: 431-434, 1991; Rosenfeld et al., Cell,68: 143-155, 1992), muscle injection (Ragot et al., Nature, 361:647-650,1993), peripheral intravenous injections (Herz and Gerard, Proc. Natl.Acad. Sci. USA, 90:2812-2816, 1993) and stereotactic innoculation intothe brain (Le Gal La Salle et al., Science, 259:988-990, 1993).

In preferred embodiments, gene therapy vectors are based uponadeno-associated virus (AAV), discussed in more detail in the Examplesbelow.

Other gene transfer vectors may be constructed from retroviruses.(Coffin, In: Virology, Fields et al., eds., Raven Press, New York, pp.1437-1500, 1990.) The retroviral genome contains three genes, gag, pol,and env. that code for capsid proteins, polymerase enzyme, and envelopecomponents, respectively. A sequence found upstream from the gag genecontains a signal for packaging of the genome into virions. Two longterminal repeat (LTR) sequences are present at the 5□ and 3□ ends of theviral genome. These contain strong promoter and enhancer sequences, andalso are required for integration in the host cell genome (Coffin,1990).

In order to construct a retroviral vector, a nucleic acid encodingprotein of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes, but without the LTR andpackaging components, is constructed (Mann et al., Cell, 33:153-159,1983). When a recombinant plasmid containing a cDNA, together with theretroviral LTR and packaging sequences is introduced into this cell line(by calcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The mediacontaining the recombinant retroviruses is then collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are capableof infecting a broad variety of cell types. However, integration andstable expression require the division of host cells (Paskind et al.,Virology, 67:242-248, 1975).

Other viral vectors may be employed as expression constructs. Vectorsderived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwaland Sugden, 1986; Coupar et al., Gene 68:1-10, 1988), adeno-associatedvirus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat andMuzycska, Proc. Natl. Acad. Sci. USA, 81: 6466-6470, 1984), and herpesviruses may be employed. They offer several attractive features forvarious mammalian cells (Friedmann, Science, 244:1275-1281, 1989;Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwichet al., J. Virol., 64:642-650, 1990).

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells also are contemplated by the present invention.These include calcium phosphate precipitation (Graham and van der Eb,Virology, 52:456-467, 1973.; Chen and Okayama, Mol. Cell. Biol.,7:2745-2752, 1987.; Rippe et al., Mol. Cell. Biol. 10: 689-695, 1990;DEAE dextran (Gopal, et al. Mol. Cell. Biol., 5:1188-1190, 1985),electroporation (Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718, 1986;Potter et al., Proc. Natl. Acad. Sci. USA, 81: 7161-7165, 1984), directmicroinjection, DNA-loaded liposomes and lipofectamine-DNA complexes,cell sonication, gene bombardment using high velocity microprojectiles,and receptor-mediated transfection (Wu and Wu, J. Biol. Chem.262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892, 1988). Some ofthese techniques may be successfully adapted for in vivo or ex vivo use.

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposome-mediated nucleic acid delivery andexpression of foreign DNA in vitro has been very successful. Wong etal., (Gene, 10:87-94; 1980) demonstrated the feasibility ofliposome-mediated delivery and expression of foreign DNA in culturedchick embryo, HeLa, and hepatoma cells. Nicolau et al., (MethodsEnzymol., 149:157-176, 1987.) accomplished successful liposome-mediatedgene transfer in rats after intravenous injection.

Pharmaceutical Compositions

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions—expression vectors, virus stocks,proteins, antibodies and drugs—in a form appropriate for the intendedapplication. Generally, this will entail preparing compositions that areessentially free of impurities that could be harmful to humans oranimals.

One generally will desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Aqueous compositions of the present invention may comprise an effectiveamount of a protein, peptide, fusion protein, recombinant phage and/orexpression vector, dissolved or dispersed in a pharmaceuticallyacceptable carrier or aqueous medium. Such compositions also arereferred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the proteins or peptides of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention are via any common route so long asthe target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal, intraarterial or intravenous injection. Suchcompositions normally would be administered as pharmaceuticallyacceptable compositions, described supra.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it is preferable to include isotonic agents,for example, sugars or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Therapeutic Agents

In certain embodiments, therapeutic agents may be attached to atargeting peptide or fusion protein for selective delivery to, forexample, non-metastatic and/or metastatic prostate cancer. Agents orfactors suitable for use may include any chemical compound that inducesapoptosis, cell death, cell stasis and/or anti-angiogenesis or otherwiseaffects the survival and/or growth rate of a cancer cell.

Regulators of Programmed Cell Death

Apoptosis, or programmed cell death, is an essential process for normalembryonic development, maintaining homeostasis in adult tissues, andsuppressing carcinogenesis (Kerr et al., 1972). The Bcl-2 family ofproteins and ICE-like proteases have been demonstrated to be importantregulators and effectors of apoptosis in other systems. The Bcl-2protein, discovered in association with follicular lymphoma, plays aprominent role in controlling apoptosis and enhancing cell survival inresponse to diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary andSklar, 1985; Tsujimoto et al., 1985). The evolutionarily conserved Bcl-2protein now is recognized to be a member of a family of relatedproteins, which can be categorized as death agonists or deathantagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli. Also, it now is apparentthat there is a family of Bcl-2 cell death regulatory proteins thatshare in common structural and sequence homologies. These differentfamily members have been shown to either possess similar functions toBcl-2 (e.g., Bcl_(XL), Bcl_(W), Bcl_(S), Mcl-1, A1, Bfl-1) or counteractBcl-2 function and promote cell death (e.g., Bax, Bak, Bik, Bim, Bid,Bad, Harakiri).

Non-limiting examples of pro-apoptosis agents contemplated within thescope of the present invention include gramicidin, magainin, mellitin,defensin, cecropin, (KLAKLAK)₂ (SEQ ID NO:1), (KLAKKLA)₂ (SEQ ID NO:2),(KAAKKAA)₂ (SEQ ID NO:3) or (KLGKKLG)₃ (SEQ ID NO:4).

Angiogenic Inhibitors

In certain embodiments the present invention may concern administrationof targeting peptides attached to anti-angiogenic agents, such asangiotensin, laminin peptides, fibronectin peptides, plasminogenactivator inhibitors, tissue metalloproteinase inhibitors, interferons,interleukin 12, platelet factor 4, IP-10, Gro-13, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2 (Regeneron),interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin,AGM-1470, platelet factor 4 or minocycline.

Proliferation of tumors cells relies heavily on extensive tumorvascularization, which accompanies cancer progression. Thus, inhibitionof new blood vessel formation with anti-angiogenic agents and targeteddestruction of existing blood vessels have been introduced as aneffective and relatively non-toxic approach to tumor treatment. (Arap etal., Science 279:377-380, 1998a; Arap et al., Curr. Opin. Oncol.10:560-565, 1998b; Ellerby et al., Nature Med. 5:1032-1038, 1999). Avariety of anti-angiogenic agents and/or blood vessel inhibitors areknown. (E.g., Folkman, In: Cancer: Principles and Practice, eds. DeVitaet al., pp. 3075-3085, Lippincott-Raven, New York, 1997; Eliceiri andCheresh, Curr. Opin. Cell. Biol. 13, 563-568, 2001).

Cytotoxic Agents

A wide variety of anticancer agents are well known in the art and anysuch agent may be coupled to a cancer targeting peptide for use withinthe scope of the present invention. Exemplary cancer chemotherapeutic(cytotoxic) agents of potential use include, but are not limited to,5-fluorouracil, bleomycin, busulfan, camptothecin, carboplatin,chlorambucil, cisplatin (CDDP), cyclophosphamide, dactinomycin,daunorubicin, doxorubicin, estrogen receptor binding agents, etoposide(VP16), farnesyl-protein transferase inhibitors, gemcitabine,ifosfamide, mechlorethamine, melphalan, mitomycin, navelbine,nitrosurea, plicomycin, procarbazine, raloxifene, tamoxifen, taxol,temazolomide (an aqueous form of DTIC), transplatinum, vinblastine andmethotrexate, vincristine, or any analog or derivative variant of theforegoing. Most chemotherapeutic agents fall into the categories ofalkylating agents, antimetabolites, antitumor antibiotics,corticosteroid hormones, mitotic inhibitors, and nitrosoureas, hormoneagents, miscellaneous agents, and any analog or derivative variantthereof.

Chemotherapeutic agents and methods of administration, dosages, etc. arewell known to those of skill in the art (see for example, the“Physicians Desk Reference”, Goodman & Gilman's “The PharmacologicalBasis of Therapeutics” and “Remington: The Science and Practice ofPharmacy,” 20th edition, Gennaro, Lippincott, 2000, each incorporatedherein by reference in relevant parts), and may be combined with theinvention in light of the disclosures herein. Some variation in dosagewill necessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Of course,all of these dosages and agents described herein are exemplary ratherthan limiting, and other doses or agents may be used by a skilledartisan for a specific patient or application. Any dosage in-betweenthese points, or range derivable therein is also expected to be of usein the invention.

Alkylating Agents

Alkylating agents are drugs that directly interact with genomic DNA toprevent cells from proliferating. This category of chemotherapeuticdrugs represents agents that affect all phases of the cell cycle, thatis, they are not phase-specific. An alkylating agent, may include, butis not limited to, nitrogen mustard, ethylenimene, methylmelamine, alkylsulfonate, nitrosourea or triazines. They include but are not limitedto: busulfan, chlorambucil, cisplatin, cyclophosphamide (cytoxan),dacarbazine, ifosfamide, mechlorethamine (mustargen), and melphalan.

Antimetabolites

Antimetabolites disrupt DNA and RNA synthesis. Unlike alkylating agents,they specifically influence the cell cycle during S phase.Antimetabolites can be differentiated into various categories, such asfolic acid analogs, pyrimidine analogs and purine analogs and relatedinhibitory compounds. Antimetabolites include but are not limited to,5-fluorouracil (5-FU), cytarabine (Ara-C), fludarabine, gemcitabine, andmethotrexate.

Natural Products

Natural products generally refer to compounds originally isolated from anatural source, and identified as having a pharmacological activity.Such compounds, analogs and derivatives thereof may be, isolated from anatural source, chemically synthesized or recombinantly produced by anytechnique known to those of skill in the art. Natural products includesuch categories as mitotic inhibitors, antitumor antibiotics, enzymesand biological response modifiers.

Mitotic inhibitors include plant alkaloids and other natural agents thatcan inhibit either protein synthesis required for cell division ormitosis. They operate during a specific phase during the cell cycle.Mitotic inhibitors include, for example, docetaxel, etoposide (VP16),teniposide, paclitaxel, taxol, vinblastine, vincristine, andvinorelbine.

Taxoids are a class of related compounds isolated from the bark of theash tree, Taxus brevifolia. Taxoids include but are not limited tocompounds such as docetaxel and paclitaxel. Paclitaxel binds to tubulin(at a site distinct from that used by the vinca alkaloids) and promotesthe assembly of microtubules.

Antibiotics

Certain antibiotics have both antimicrobial and cytotoxic activity.These drugs also interfere with DNA by chemically inhibiting enzymes andmitosis or altering cellular membranes. These agents are not phasespecific so they work in all phases of the cell cycle. Examples ofcytotoxic antibiotics include, but are not limited to, bleomycin,dactinomycin, daunorubicin, doxorubicin (Adriamycin), plicamycin(mithramycin) and idarubicin.

Miscellaneous Agents

Miscellaneous cytotoxic agents that do not fall into the previouscategories include, but are not limited to, platinum coordinationcomplexes, anthracenediones, substituted ureas, methyl hydrazinederivatives, amsacrine, L-asparaginase, and tretinoin. Platinumcoordination complexes include such compounds as carboplatin andcisplatin (cis-DDP). An exemplary anthracenedione is mitoxantrone. Anexemplary substituted urea is hydroxyurea. An exemplary methyl hydrazinederivative is procarbazine (N-methylhydrazine, MIH). These examples arenot limiting and it is contemplated that any known cytotoxic, cytostaticor cytocidal agent may be attached to targeting peptides andadministered to a targeted organ, tissue or cell type within the scopeof the invention.

Cytokines and Chemokines

In certain embodiments, it may be desirable to couple specific bioactiveagents to one or more targeting peptides for targeted delivery to anorgan, tissue or cell type. Such agents include, but are not limited to,cytokines and/or chemokines.

The term “cytokine” is a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Examplesof cytokines are lymphokines, monokines, growth factors and traditionalpolypeptide hormones. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-alpha. and -beta; mullerian-inhibiting substance;mouse gonadotropin-associated peptide; inhibin; activin; vascularendothelial growth factor; integrin; thrombopoietin (TPO); nerve growthfactors such as NGF-.beta.; platelet-growth factor; transforming growthfactors (TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growthfactor-I and -II; erythropoietin (EPO); osteoinductive factors;interferons such as interferon-α, -β, and -γ; colony stimulating factors(CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF(GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1,IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, LIF, G-CSF,GM-CSF, M-CSF, EPO, kit-ligand and or FLT-3, angiostatin,thrombospondin, endostatin, tumor necrosis factor and LT. As usedherein, the term cytokine includes proteins from natural sources or fromrecombinant cell culture and biologically active equivalents of thenative sequence cytokines.

Chemokines generally act as chemoattractants to recruit immune effectorcells to the site of chemokine expression. It may be advantageous toexpress a particular chemokine gene in combination with, for example, acytokine gene, to enhance the recruitment of other immune systemcomponents to the site of treatment. Chemokines include, but are notlimited to, RANTES, MCAF, MIP1-alpha, MIP1-Beta, and IP-10. The skilledartisan will recognize that certain cytokines are also known to havechemoattractant effects and could also be classified under the termchemokines.

Dosages

The skilled artisan is directed to “Remington: The Science and Practiceof Pharmacy,” 20th edition, Gennaro, Lippincott (2000). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and general safety and purity standards as required by theFDA Office of Biologics standards.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Biopanning with Phage Display Libraries Using Human Patients

Certain of the methods and compositions of the present invention concernidentification of targeting peptides for human organs, tissues or celltypes by in vivo biopanning. Generally, protocols used in animalsubjects, such as mice, are not suited for humans. Further, ethicalconsiderations play a large role in human protocols. The following novelmethods are preferred for use with humans, although the skilled artisanwill realize that variations on the methods and compositions disclosedherein may be used within the scope of the present invention.

Human Preparation

Patients were selected for the protocol according to inclusion andexclusion criteria. Inclusion criteria include: (1) patient legallydeclared brain dead or terminal wean patient; (2) approval of attendingand/or treating physicians; and (3) approved written informed consentform signed by the patient's legally responsible family member.Exclusion criteria were: (1) the absence of a responsible family member;(2) HIV positive patient; (3) patient with active tuberculosisinfection; (4) acute or chronic hepatitis B or C infections; or (5)patient was a potential organ transplant donor. In preferredembodiments, the patient was not on antibiotics for at least theprevious 6 hrs, preferably the last 24 hrs, in order to avoiddetrimental effects on the bacterial hosts used to propagate the phageused for the peptide display library.

After informed consent and before the patient was prepared for theprocedure, relatives of the patient were asked to leave the room thepatient was in. The patient had a well running IV line (preferablycentral) with nothing but saline running through the channel ofapplication of the phage library. Personnel required for the procedurewere notified (i.e., intervention radiologist, internist, surgeon,nurse, possibly neurologist or neuroradiologist). Materials needed forbiopsies were collected: bone marrow aspiration needle, lumbar puncturekit, skin biopsy kit, materials for taking biopsies of any organ, tissueor cell type used for targeted peptide identification, such as liver,fat and tumor, materials for transabdominal prostate biopsy, 50 mlsyringe with 40 ml saline for blood sample, 10 ml tube containingheparin and 10 ml serum collection tube to draw blood sample for labtests. Before phage library injection, blood samples were drawn forroutine screening of liver function, bicarbonate, electrolytes and bloodcount, unless test results from the day of the injection were available.

In the laboratory, 120 large dishes with LB-tet/kan agar as well as 200regular LB tet/kan plates (100 mm) were prepared (tetracyclineconcentration=40 μg/ml, kanamycin concentration=50 μg/ml). E. coli K91kan were grown in 10 independent tubes, each containing 10 ml TB mediumplus supplements. Growth of bacteria was started approx. 15-60 min priorto beginning the biopsies. About 10¹⁴ TU of the (preferably primary)phage library were diluted in 200 ml ringer lactate at room temperatureand aspirated under clean but not necessarily sterile conditions intofour 50 ml syringes. LB-tet/kan dishes or plates were warmed in a 37° C.incubator. One liter of LB medium containing 0.2 μg/ml tet and 100 μg/mlkan was warmed in the waterbath at 37° C. One liter LB medium containing40 μg/ml tet and 100 μg/ml kan was warmed to 37° C. and 8 more literswere prepared at room temperature. Thirty glass grinders A and B size aswell as suitable glass tubes were autoclaved. Three 50 ml Falcon tubeswere prepared for each of the organs for which biopsies were to betaken. Tubes were filled with 10 ml DMEM-PI-DMEM containing PMSF (1 mM),aprotinin (20 μg/ml) and leupeptin (1 μg/ml)- and put on iceapproximately 15 minutes before tissue collection. For each of the 4teams taking over in the lab after the tissue samples were collected,one autoclaved set of surgicals (i.e., at least one forceps and one pairof scissors and a scalpel) were prepared in order to trim, divide ormince organ samples.

Phage Library Injection

All drugs running through the intended port of application of the phagelibrary were discontinued during library injection. If possible withoutcompromising the patient's hemodynamic stability, all IV drugs runningthrough different ports were discontinued during library injection aswell. A running saline infusion ensured that the IV line for the libraryinjection was open and was left running during the injection.

The 200 ml library solution was manually injected over a period of 10minutes while monitoring and protocoling the patient's vital functionssuch as breathing (if not mechanically ventilated), heart rate and bloodpressure. The injection was stopped any time the running saline infusionstopped dripping, indicating obstruction of the line. Fifteen minutesafter beginning the injection, tissue sample collection (biopsies) wasinitiated. Biopsy sites included bone marrow aspirate, liver, prostate,skin, skeletal muscle, tumor (if applicable), adipose tissue, blood (aspositive control), blood (for red/white blood cells) and cerebral-spinalfluid (CSF).

The samples were taken under very clean if not sterile conditions toreduce contamination with bacteria. To the extent possible, thedifferent samples were taken simultaneously. For small samples,triplicate biopsies were preferred. The time elapsed between beginningof injection and the collection of a particular tissue sample wasrecorded. Tissue samples were placed in the prepared 50 ml tubescontaining DMEM-PI and stored on ice. For bone marrow, a regulardiagnostic sample (undiluted into a syringe with heparin) was taken inaddition to the samples diluted in 40 ml saline to confirm aspiration ofbone marrow as opposed to blood. If needed, all IV drugs, includingantibiotics, were continued after removal of tissue samples.

All organ samples that were not taken in triplicate were divided underclean conditions to obtain three different pieces of tissue. The threesamples of each organ were handled as follows. One piece was storedat—80° C. as a backup. One piece was forwarded to thehistology/pathology department to cut cryosections (or to make smearsfor bone marrow) and perform HE staining (Pappenheim staining for bonemarrow) as well as phage staining to confirm that the samples containedthe organ of interest. In some cases the histology sample was divided intwo—one for regular HE staining and one for LCM (laser capturemicroscopy) or LPC (laser pressure catapulting). The last of the threeoriginal pieces was processed for bacterial infection to recover phage.

After freezing of backup tissue and saving material for pathology,samples for phage rescue were weighed. Samples were kept on ice at alltimes. Sample was transferred to 1 ml DMEM-PI in a glass tube andhomogenized with a grinder. Some organs such as bone marrow, blood, orCSF do not require homogenization, whereas other organs like muscle needto be minced before they can be efficiently homogenized. Lysis oferythrocytes for blood samples was preferred. Homogenized samples weretransferred to autoclaved 2 ml Eppendorf tubes.

Tissue samples were washed 3 times with ice cold DMEM-PI containing 1%BSA by mixing the tissue with DMEM-PI and vortexing for 30 seconds.After centrifugation at 4,000 rpm for 3 min, supernatant was carefullydiscarded, leaving the tissue pellet undisturbed. A small amount ofmedium was left on the surface of the pellet. Samples were vortexedagain for 30 seconds before adding more medium to facilitateresuspension of the tissue. After adding 1.5 ml of DMEM-PI plus BSA thesamples were centrifuged again. When processing multiple samples, thetissues were kept on ice at all times.

After 3 washes, the pellet was briefly vortexed and the dissolved pelletwas warmed briefly to 37° C. before adding bacteria. The washed tissuesamples were incubated with 1.5 ml of competent K91-kan bacteria (OD₆₀₀0.2 in 1:10 dil.) for one hour at room temperature, then transferred toFalcon tubes containing 10 ml of LB medium with 0.2 μg/ml tetracycline.After 20 min at RT, multiple aliquots were plated on LB tet/kan platesor dishes containing 40 μg/ml of tetracycline and 100 μg/ml kanamycin.The following quantities (per organ sample) were plated: 2 dishes with 3ml; 2 dishes with 1 ml; 3 dishes with 300 μl; 3 dishes with 100 μl; 3dishes with 30 μl.

The beads that were used for plating were passed on to two subsequent 10cm LB tet/kan plates to recover every potentially phage infectedbacterial clone that might be trapped on the bead surface. Dishes wereincubated overnight at 37° C.

The remaining 2-3 ml of infected culture (including the homogenizedtissue) was transferred to 10 ml of LB medium containing 40 μg/mltetracycline and 100 μg/ml kanamycin (LB tet/kan) and shaken at 37° C.for 2 hr. This approximately 12 ml culture was transferred to 1 liter LBtet/kan and grown overnight in a 37° C. shaker.

The next day, phage were rescued from the bulk amplified bacterialculture according to standard protocols and saved for a potential secondround of in vivo selection. From the plates/dishes in the incubator,1500 well separated colonies were picked for each organ plated andtransferred to 96 well plates containing 20 μl PBS/well for sequencing.This assumed a readout of about 2 out of 3 picked colonies to obtain1000 sequences.

After picking 1500 colonies, the remainder of colonies on thedishes/plates were grown in 1000 ml LB tet/kan overnight in the 37° C.shaker. Then phage were harvested as before for a second round ofselection. Alternatively, the plates were stored in the refrigerator and1000-2000 individual colonies grown at a time. Alternatively, theremainder of colonies were transferred to PBS and stored frozen toinfect and amplify as needed.

Numerous non-limiting examples of human organ, tissue or cell typeselective targeting peptides have been identified by in vivo biopanningusing the present methods, as disclosed below.

Example 2 Mapping the Human Vasculature by In vivo Phage Display

The in vivo selection method discussed above was used to screen a phagelibrary in a human subject. A pattern recognition analysis program wasused to survey 47,160 tripeptide motifs within peptides that localizedto the human bone marrow, fat, skeletal muscle, prostate, or skin. Theresults of this large-scale screening indicated that the distribution ofcirculating peptide motifs to different organs is non-random.High-throughput analysis of peptide motifs enriched in individualtissues revealed similarities to sequences present in candidate ligandsfor differentially expressed vascular receptors.

These data represent a major step towards the construction of aligand-receptor map of human vasculature and may have broad implicationsfor the development of targeted therapies. Many therapeutic targets maybe expressed in very restricted—but highly specific andaccessible—locations in the vascular endothelium. Potential targets forintervention may be overlooked in high-throughput DNA sequencing or ingene arrays because these approaches do not usually take into accountcellular location and anatomical, and functional context. The human invivo phage display screening methods disclosed herein are uniquelysuited to identification of naturally occurring ligand-receptor pairsthat may provide the basis for highly selective therapies againstvarious disease states.

Materials and Methods

A 48 year-old male Caucasian patient who had been diagnosed withWaldenström macroglobulinemia (a B cell malignancy) was previouslytreated by splenectomy, systemic chemotherapy (fludarabine,mitoxantrone, and dexamethasone), and immunotherapy (anti-CD20monoclonal antibody). In the few months prior to his admission, thedisease became refractory to treatment and clinical progression occurredwith retroperitoneal lymphadenopathy, pancytopenia, and marked bonemarrow infiltration by tumor cells. The patient was admitted withmassive intracranial bleeding secondary to thrombocytopenia. Despiteprompt craniotomy and surgical evacuation of a cerebral hematoma, thepatient remained comatose with progressive and irreversible loss ofbrainstem function until the patient met the formal criteria forbrain-based determination of death, as evaluated by an independentclinical neurologist. Because of his advanced cancer, the patient wasrejected as transplant organ donor. After surrogate written informedconsent was obtained from the legal next of kin, in vivo phage displaywas performed.

In Vivo Phage Display

A large-scale preparation of a CX₇C(C, cysteine; X, any amino acidresidue) phage display random peptide library was optimized to createthe highest possible insert diversity (Pasqualini et al., 2000). Thediversity of the library was about 2×10⁸ and its final titer was about10¹² transducing units (TU)/ml. For biopanning with human subjects, useof a large-scale phage display library (diversity about 2×10⁸) isadvantageous compared to the smaller scale libraries used in mousestudies. Short-term intravenous infusion of the phage library (a totaldose of 10¹⁴ phage TU suspended in 100 ml of saline) into the patientwas followed by multiple representative tissue biopsies. Prostate andliver samples were obtained by needle biopsy under ultrasonographicguidance. Skin, fat tissue, and skeletal muscle samples were obtained bysurgical excision. Bone marrow needle aspirates and core biopsies werealso obtained. Histopathological diagnosis was determined by examinationof frozen sections processed from tissues obtained at the bedside.

Triplicate samples were processed for host bacterial infection, phagerecovery, and histopathological analysis. In brief, tissues wereweighed, ground with a glass Dounce homogenizer, suspended in 1 ml ofDulbecco Modified Eagle's medium (DMEM) containing proteinase inhibitors(DMEM-prin; 1 mM PMSF, 20 μg/ml aprotinin, and 1 μs/ml leupeptin),vortexed, and washed three times with DMEM-prin. The human tissuehomogenates were incubated with 1 ml of host bacteria (log phase E. coliK91kan; OD₆₀₀ ˜2). Aliquots of the bacterial culture were plated ontoLuria-Bertani agar plates containing 40 μg/ml tetracycline and 100 μg/mlof kanamycin. Plates were incubated overnight at 37° C. Bacterialcolonies were processed for sequencing of phage inserts recovered fromeach tissue and from unselected phage library. Human samples werehandled with universal blood and body fluid precautions.

Statistical Analysis

A high-throughput character pattern recognition program (M.D. AndersonCancer Center, Biostatistics, Houston, Tex.) was developed to automatethe analysis of the peptide motifs derived from phage screenings. Byusing SAS (version 8, SAS Institute) and Perl (version 5.0), the programconducts an exhaustive amino acid residue sequence count and tracks therelative frequencies of N distinct tripeptide motifs representing allpossible n₃ overlapping tripeptide motifs in both directions (N<<n₃).This was applied for phage recovered from each target tissue and for theunselected CX₇C random phage display peptide library.

With “p” defined as the probability of observing a particular tripeptidemotif under total randomness, and q=1−p, the probability of observing Ksequences characterized as a particular tripeptide motif out of n₃ totaltripeptide motif sequences is binomial (n₃, p). That probability may beapproximated by the formula: p_(K)=Ψ[(k+1)/sqrt(n₃ pq)]−Ψ[k/sqrt(n₃pq)], where Ψ is the cumulative Gaussian probability. The value p_(K)may be treated as a P-value in testing for total randomness of observingexactly K sequences of a particular tripeptide motif. However, this testrequires exact knowledge of the true value of p, which it is difficultto obtain in practice.

In order to identify the motifs that were enriched in the screening, thecount for each tripeptide motif within each tissue was compared with thecount for that tripeptide motif within the unselected library. Startingfrom a CX₇C peptide insert, counts were recorded for all overlappinginterior tripeptide motifs, subject only to reflection and single-votingrestrictions. No peptide was allowed to contribute more than once for asingle tripeptide motif (or a reflected tripeptide motif). Each peptidecontributed five tripeptide motifs. Tripeptide motif counts wereconditioned on the total number for all motifs being held fixed within atissue. The significance of association of a given allocation of countswas assessed by Fisher's exact test (one-tailed). Results wereconsidered statistically significant at P<0.05. In summary, to test forrandomness of distribution, the relative frequencies of a particulartripeptide motif from each target was compared to the frequencies of themotifs from the unselected library. This approach is a large-scalecontingency table association test.

Results

Phage localizing to human prostate tissue exhibited targeting peptidesequences as disclosed in Table 3, minus the terminal cysteine residueson each end of the peptides.

TABLE 3 Human Prostate Targeting Peptides GRRAGGS (SEQ ID NO: 5) TRRAGGG(SEQ ID NO: 6) SRAGGLG (SEQ ID NO: 7) SYAGGLG (SEQ ID NO: 8) DVAGGLG(SEQ ID NO: 9) GAGGLGA (SEQ ID NO: 10) GAGGWGV (SEQ ID NO: 11) AGGTFKP(SEQ ID NO: 12) LGEVAGG (SEQ ID NO: 13) GSNDAGG (SEQ ID NO: 14) YRGIAGG(SEQ ID NO: 15) AGGVAGG (SEQ ID NO: 16) GGLAGGF (SEQ ID NO: 17) LLAGGVL(SEQ ID NO: 18) LVVSAGG (SEQ ID NO: 19) RTQAGGV (SEQ ID NO: 20) AGGFGEQ(SEQ ID NO: 21) AGGLIDV (SEQ ID NO: 22) AGGSTWT (SEQ ID NO: 23) AGGDWWW(SEQ ID NO: 24) AGGGLLM (SEQ ID NO: 25) VAAGGGL (SEQ ID NO: 26) LYGAGGS(SEQ ID NO: 27) CALAGGC (SEQ ID NO: 28) IGAGGVH (SEQ ID NO: 29)

To determine the distribution of the peptide inserts homing to specificsites after intravenous administration, the relative frequencies ofevery tripeptide motif from prostate tissue were compared to thefrequencies from the unselected library. The 1,018 phage insertsrecovered from representative samples of prostate and from theunselected library were analyzed. Tripeptide motifs were chosen for thephage insert analysis because three amino acid residues appear toprovide the minimal framework for structural formation andprotein-protein interaction (Vendruscolo et al., 2001). Examples ofbiochemical recognition units and binding of tripeptide ligand motifs toreceptors include RGD (Ruoslahti, 1996), LDV (Ruoslahti, 1996), and LLG(Koivunen et al., 2001) to integrins, NGR (Pasqualini et al., 2000) toaminopeptidase N/CD13, and GFE (Rajotte and Ruoslahti, 1999) to membranedipeptidase.

Each phage insert analyzed contained seven amino acid residues andcontributed five potential tripeptide motifs. Comparisons of the motiffrequencies in prostate tissue are shown in Table 4. The AGG (SEQ IDNO:30) motif was found only in prostate homing phage, while the othertripeptide motifs were all found in at least one other tissue. Table 4lists motifs occurring in peptides isolated from prostate but not fromthe unselected phage library (Fisher's exact test, one-tailed; P<0.05).

TABLE 4 Peptide Motifs Isolated from Prostate byIn Vivo Phage Display in Humans Motif Motif Frequency P-value AGG(SEQ ID NO: 30) 2.5 0.0340 EGR (SEQ ID NO: 31) 1.0 0.0185 GER(SEQ ID NO: 32) 0.9 0.0382 GVL (SEQ ID NO: 33) 2.3 0.0079

The ClustalW program (European Molecular Biology Laboratory; EMBL) wasused to analyze the original cyclic phage peptide inserts of seven aminoacid residues containing the tripeptide motifs. The analysis revealedfive to six residue motifs that were shared among multiple peptidesisolated from prostate (Table 5), including RRAGGS (SEQ ID NO:34) andRRAGG (SEQ ID NO:35). On-line databases were searched for each of themotifs (including BLAST, SWISSPROT, PROSITE, PRODOM, and BLOCKS) throughthe NCBI website (http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp).These motifs are likely to represent sequences present in circulatingligands (either secreted proteins or surface receptors in circulatingcells) that home to vascular receptors in prostate. Candidate humanproteins potentially mimicked by the selected peptide motifs arepresented in Table 5.

TABLE 5 Examples of human proteins potentiallymimicked by peptide motifs Extended Human Protein Accession motifprotein description number Prostate RRAGGS Interleu-  cytokine NP_000632(SEQ ID NO: 34) kin 11 RRAGG Smad6 Smad  AAB94137 (SEQ ID NO. 35) familymember

Table 5 shows sequences corresponding to regions of 100% sequenceidentity between the peptide selected and the candidate protein. Theidentified homologous proteins may represent natural ligands for thehuman receptors that bound targeting phage. For example, interleukin 11has been reported to interact with receptors within endothelium andprostate epithelium (Mahboubi et al., 2000). IL-11 may be mimicked by atargeting peptide recovered from the prostate (Table 5). These resultswere confirmed by in situ staining, using polyclonal antibodies againstIL-11 receptor alpha. IL-11 is a cytokine that is apparently mimicked bythe peptide motif RRAGGS (SEQ ID NO:34), a human prostate targetingpeptide. This suggests that the IL-11 receptor alpha (IL-11Rα) should beoverexpressed in prostate blood vessels. Studies with cultured cellshave indicated that IL-11 interacts with receptors in endothelium andprostate epithelium (Mahboubi et al., 2000; Campbell et al., 2001).However, expression of IL-11Rα in prostate blood vessels has notpreviously been examined.

Immunostaining of prostate thin sections with antibodies against IL-11Rαshowed that IL-11Rα is present in the luminal prostate epithelium and inprostate blood vessels (not shown). This result validates the humanbiopanning results and shows that the presence of cell surface receptorsidentified by targeting peptide binding can be confirmed by antibodiesagainst the receptor protein.

A considerable advantage of the present method is that the selectedtargeting peptides bind to native receptors, as they are expressed invivo. Even if a ligand-receptor interaction is mediated through aconformational (rather than a linear) epitope, it is still possible toselect binders in the screening. As it is difficult to ensure thattransmembrane proteins expressed by recombinant systems (such as inprotein arrays) maintain the correct structure and folding afterpurification in vitro, peptides selected in vivo are likely to be moresuitable to clinical applications, such as identification of novelinhibitors or activators of native receptor proteins.

The skilled artisan will realize that the prostate-targeting peptidesequences identified in the present Example will be of use for numerousapplications within the scope of the present invention, including butnot limited to targeted delivery of therapeutic agents or gene therapy,in vivo imaging of normal or diseased organs, tissues or cell types,identification of receptors and receptor ligands in organs, tissues orcell types, and therapeutic treatment of human diseases, such as benignprostatic hyperplasia (BPH) and/or prostate cancer.

Example 3 The IL-11Receptor as a Therapeutic and Diagnostic Target inCancer

The preceding Example identified prostate-targeting motifs (RRAGGS, SEQID NO:34 and RRAGG, SEQ ID NO:35) in normal human prostate tissue. Thehomology of the RRAGGS (SEQ ID NO:34) motif with human IL-11 suggeststhat the native prostate receptor for binding of RRAGGS (SEQ ID NO:34)may be the IL-11 receptor. The present Example determined whether theIL-11 receptor could be targeted in prostate cancer, includingmetastatic prostate cancer.

To test the tissue specificity of the IL-11 peptide mimic, a phageoverlay assay was developed to evaluate receptor-ligand interactions intissue sections, using the motif RRAGGS (SEQ ID NO:34) (Arap et al.,Nature Med. 8:121-127, 2002). Phage overlay on human tissue sectionsshowed that the prostate-homing phage displaying an IL-11 peptide mimicspecifically bound to the endothelium and epithelium of normal prostate,but not to control organs, such as skin (data not shown). In contrast, acontrol phage that localized to skin tissue, displaying the motif HGGVG(SEQ ID NO:36), did not bind to prostate tissue (not shown). However,the control phage specifically recognized blood vessels in the skin (notshown).

The immunostaining pattern obtained with an antibody against humanIL-11Rα (IL-11 receptor alpha) on normal prostate tissue wasindistinguishable from that of a CGRRAGGSC (SEQ ID NO:37)-displayingphage overlay (not shown). In contrast, a control antibody showed nostaining in prostate tissue (not shown). These findings wererecapitulated in multiple tissue sections obtained from severaldifferent patients (Arap et al., 2002).

Using a ligand-receptor binding assay in vitro, the interaction of theCGRRAGGSC (SEQ ID NO:37)-displaying phage with immobilized IL-11Rα wasdemonstrated at the protein-protein level (not shown). RecombinantIL-11Rα, vascular endothelial growth factor receptor-1 (VEGFR1) orleptin receptor (OB-R) were incubated with phage displaying theCGRRAGGSC (SEQ ID NO:37) peptide. VEGFR1 was used as a representativevascular receptor, while OB-R was used because it shares a co-receptorwith IL-11Rα. An unrelated phage clone displaying the peptide CRVDFSKGC(SEQ ID NO:38) and insertless fd-tet phage were used as controls. Onlythe IL-11Rα receptor protein exhibited a significant amount of bindingto CGRRAGGSC (SEQ ID NO:37)-phage (not shown). Neither OB-R nor VEGFR1showed binding to CGRRAGGSC (SEQ ID NO:37)-phage above control levels(not shown). Neither of the control phage exhibited selective binding toIL-11Rα (not shown). Binding of CGRRAGGSC (SEQ ID NO:37)-phage toIL-11Rα was specific, since it was inhibited by the native IL-11 ligandin a concentration-dependent manner (not shown). Close to 100%inhibition of CGRRAGGSC (SEQ ID NO:37)-phage binding was observed at apeptide concentration of about 0.1 nM (not shown). These observationswith normal prostate tissues were followed by an examination of theexpression of IL-11Rα in tumors, as discussed in the present Example.IL-11R expression was found to be upregulated in human prostate cancer(see below).

Characteristics of IL-11Receptor

IL-11Rα is a member of the 130-dependent family of proteins, along withreceptors for IL6, oncostatin M, leukemia inhibitory factor, andcilliary neurotrophic factor (Du and Williams, Blood 89:3897-3908,1997). IL-11 initiates signaling via binding to the unique IL-11Rαchain, The complex of IL-11 and IL-11Rα then binds to and inducesclustering of gp130, leading to the activation of associated Januskinases (JAKs) and translocation to the nucleus of the signaltransducers and activators of transcription (STAT) proteins 3 and 1(Lutticken et al., Science 263:89, 1994; Campbell et al., Am. J. Pathol.158:25-32, 2001). STAT3 has been reported to be constitutively activatedin prostate cancer (Ni et al., J. Urol. 167:1859-62, 2002). IL-11Rαexpression was reported to be increased in primary prostatic carcinomacompared to non-malignant prostate tissue (Campbell et al., 2001). Noprevious reports have characterized IL-11Rα expression in metastaticcancer.

Other signaling systems that may be activated by IL-11Rα include MAPkinase, and the ribosomal S6 protein kinase pp90rsk, src-family tyrosinekinases including p60src and p62yes, and phosphatidylinositol-3 kinase.IL-11Rα has been characterized on human solid tumors such as breast,colon, ovary, and melanoma (Douglas et al., Oncogene 14:661-69, 1997;Gupta et al., Proc. Am. Assn. Cancer Res. 38:554, 1997; Paglia et al.,J. Interf. Cytokine Res., 15:455-460, 1995; Campbell et al, Gynecol.Oncol. 80:121-27, 2001), although its functional role and prognosticsignificance were unknown.

Distribution of IL-11Rα in Primary and Metastatic Prostate Cancer

Immunohistochemical (IHC) analysis was performed to examine thedistribution of IL-11Rα in primary prostate cancer and metastaticprostate cancer. The present Example represents the first report ofIL-11Rα distribution in metastatic cancer of any kind. Normal tissuesfrom different areas in the prostate were also examined. Tissues from 99archival formalin-fixed paraffin-embedded human primary and metastaticprostate cancers and the corresponding adjacent non-neoplastic tissueswere obtained from 90 patients and evaluated. Samples consisted of 81primary adenocarcinomas (71 androgen-dependent [AD] obtained fromradical prostatectomy without prior treatment, and 10androgen-independent [AI] obtained either from radical prostatectomy,cystoprostatectomy, or pelvic exenteration) and 18 lymph node and bonemetastases and were selected to reflect: 1) stages in prostate cancerprogression; 2) different Gleason scores; 3) hormonal dependence: AD andAI tumours; and 4) zonal origin: peripheral zone and transition zone.Additional blocks from the same specimens, including benign prostatictissue from peripheral zone, transition zone, and central zone, and theseminal vesicle/ejaculatory duct, were included when available.

The samples were stained within two weeks of cutting to minimize loss ofimmunoreactivity. Four-μm sections were conventionally deparaffinizedand rehydrated, blocked for endogenous peroxidases, antigen-retrieved ina microwave oven by treatment with EDTA solution (pH 8.0; Zymed, SanFrancisco, Calif.), and biotin and protein blocked (both from DAKOCorp., Carpinteria, Calif.). Incubation with anti-human IL-11Rα K-20(1:15 for 45 minutes at room temperature; Santa Cruz Biotechnology,Santa Cruz, Calif.) followed. The LSAB+ kit (DAKO) was used forimmunostaining and development. All sections from each specimen werefrom the same staining run to avoid interassay variability.

Competition experiments with the antigenic peptide (5:1 w/w absorption;Santa Cruz) were performed to confirm specificity. Paraffin sections ofthe HeLa cell line were used as immunopositive controls. Negativecontrols included omission of the primary antibody, and substitution ofprimary antibody with non-immune goat serum at equivalent immunoglobulinconcentration. Endothelial cells were immunostained by JC/70A(anti-CD31, DAKO) monoclonal antibody. IL-11Rα staining was evaluatedboth in tumour and non-tumour tissues, including pathologic conditionsas benign prostatic hyperplasia (BPH) and transitional metaplasia, andhigh-grade prostatic intraepithelial neoplasia (PIN).

Positive cases were defined by the presence of cytoplasmic staining, asseen in the positive controls. Intensity in benign and malignant tissueswas scored as 0 (negative), 1+ (weak), 2+ (moderate), or 3+ (strong).IL-11Rα expression in benign glands was generally observed in the basalcell compartment with/without staining of the luminal cells. Weevaluated benign glands from 1+ (no/weak luminal cell staining) to 3+,taking into account then the highest intensity of staining in theluminal compartment, and compared this score with the most prevalent oneobserved in the cancerous tissue from the corresponding area. Due toheterogeneity in intensity among and within tumour samples, a totalimmunostaining score was calculated as the sum of the products ofpercentage of cells (in 10% units) per intensity level (up to a maximumscore of 300) to evaluate differences in expression among cancerousspecimens. All analyses were done with S-PLUS 2000 (Math Soft, Inc.).

TABLE 6 Clinical and histopathological characteristics and IL11Rαexpression Number Median score Specimen of cases (range)* p Normalprostate Peripheral zone 62 1 + (1-2) NS§ Transition zone 51 1 + (1-2)Central zone 40 1 + (1-2) Seminal vesicle/ 43/3 2 + (2-3)/ — EjaculatoryDuct 2 + (2) Benign pathologic conditions Benign prostatic hyperplasia15 1 + (1-2) — Stromal nodule 2 1 + (1-2) — Atrophy 10 2 + (1-2) —Transitional metaplasia 18 2 + (1-2) — Prostatic intraepithelial 23 2 +(1-3) — neoplasia (PIN) Primary prostate cancer Androgen-dependent 712 + (1-3)/ — 180 (50-290) Zonal origin Peripheral zone 55 190 (50-290)0.0003|| Transition zone 16 135 (50-250) Gleason score†  ≦7 (3 + 4) 26150 (50-260) 0.004¶  ≧7 (4 + 3) 38 200 (100-290) Pathological stage†pT₂₋pT_(3a) 42 175 (50-290) 0.046¶ pT_(3b)-pT_(any)PN₁ 22 210 (100-280)PSA (ng/mL)†  <10 48 180 (50-280) NS¶ ≧10 14 200 (100-290)Androgen-independent 10 250 (80-300) — Metastatic prostate cancer Lymphnodes Androgen-dependent 4 235 (200-290) NS|| Androgen-independent 8 235(190-300) Bone 6 270 (140-290) — NS = non-significant. *Categories 1+-3+were used for evaluation of benign prostatic tissues and comparison toprostatic intraepithelial neoplasia and primary prostate cancer. Acombined intensity per percentage of immunostained tumour cells scoringsystem was used to evaluate differences in expression among cancerousspecimens (see text). †Only the predominant tumour focus in each casewas considered (64/71 cases). §Wilcoxon signed rank test. ||Mann-Whitneyrank sum test. ¶Spearman correlation test.

No differences were observed in IL-11Rα expression between normal glandsin the different prostatic areas (Table 6). Some background, distinct toa frequent stromal staining, was observed in the epithelium of seminalvesicles and ejaculatory ducts. Expression in PIN and AD samplesexamined was significantly higher than in their benign counterparts fromthe same areas (p<0.0001 in both cases, Wilcoxon signed rank test), butno differences were observed between PIN and AD (p=0.5, signed ranktest). Among primary AD specimens, IL-11Rα immunoreactivity wasincreased in cancers from the peripheral vs. transition zone (p=0.0003),in Gleason ≧7 (4+3) vs. Gleason ≦7 (3+4) (p=0.004), and, moremarginally, in pT_(3b)−pT_(any)pN₁ tumours vs. pT₂−pT_(3a) (p=0.046)(Table 6).

Primary AI specimens showed a more homogeneous pattern of staining, withmore than 80% cells displaying moderate/strong intensity in 80% of thesamples. However, no significant increase in expression was observed inAI vs. AD cases matched by Gleason score (p=0.15, rank-sum test), likelybecause of the small number of samples. Expression in 6 regional (4 ADand 2 AI) and 6 distant lymph node metastases (6 AI) was also intense ina high percentage of tumour cells. Cancer cells displayed a homogeneousmoderate to strong intensity of staining in 5 out of 6 specimens frombone metastases (all AI). Both osteoblasts and osteoclasts stainedmoderately, and were used as internal positive controls. Interestingly,blood vessels in bone and lymph node metastases and in primary caseswith previous treatment, showed an occasionally striking IL11Rαimmunoreactivity that was confirmed by CD31 staining on consecutiveslides, as opposed to a more random pattern in the other benign andmalignant tissues analysed.

The results show that IL-11Rα expression correlates with tumorprogression (FIG. 1 and FIG. 2). FIG. 1 shows localization of IL-11Rα inbenign prostate glands. Normal prostate glands in the peripheral (FIG.1A) or central (FIG. 1B) zones showed predominantly nuclear staining ofthe basal and luminal cell layers.

FIG. 2 illustrates IHC staining for IL-11Rα in primary androgendependent prostate cancer of low (FIG. 2A), intermediate (FIG. 2B) andhigh (FIG. 2C) Gleason grade prostate tumors. FIG. 2A shows IL-11Rαdistribution in a Gleason score 6 prostate adenocarcinoma (homogeneous2+ staining). FIG. 2B shows IL-11Rα distribution in prostate carcinoma(arrowheads) (1+ and 2+ staining). The prostate carcinoma exhibitedelevated staining for IL-11Rα compared to adjacent luminal cells ofbenign prostate (arrows). Strong (3+) staining for IL-11Rα was observedin high-grade prostate adenocarcinoma (FIG. 2C). FIG. 2D shows thatbenign prostate glands from the peripheral zone, containing a fewneoplastic acini, exhibited little or no staining for IL-11Rα comparedto prostate cancer.

IL-11Rα expression was strongly up-regulated in metastatic prostatecancer (FIG. 3). FIG. 3A shows strong homogenous (3+) staining inprostate cancer that had metastasized to bone. A higher powermagnification of the same sample shows 2+ and 3+ staining in tumor cells(FIG. 3B). FIG. 3C shows that small blood vessels around tumor nodulesin the bone matrix also exhibited strong staining for IL-11Rα. CD31staining of the same sample (FIG. 3D) confirmed the endothelial cellreactivity of the IL-11Rα IHC staining. A high-grade,androgen-independent primary prostate tumor also exhibited strong (3+)staining for IL-11Rα (FIG. 3E). A negative control of benign prostatetissue from the same area as FIG. 3B exhibited little or no staining forIL-11Rα (FIG. 3F). FIG. 33 shows the distribution of IL-11Rα expressionin primary androgen-dependent prostate carcinoma by immunohistochemicalscore, according to Gleason grade and pathological stage.

IL-11Rα expression was examined in blood vessels of prostate tissuesamples. Although staining was observed in some prostate blood vessels,it was not observed in others. A sub-group of cases displayed a strongerand more consistent staining in blood vessels. The majority of suchcases were androgen-independent, including both primary and metastaticandrogen-independent tumors (17 of 24 AI cases). Blood vessel stainingin AI tumors could result from either a shift in hormonal dependence orexposure to previous treatment. No common modality of treatment wasobserved in such cases, with the exception of hormone ablation. A fewcases had been treated with radiotherapy and the rest with differenttype of chemotherapy. In some cases the systemic treatments had beenadministered a long time before sample analysis for IL-11Rα expression.It is concluded that androgen independence is correlated with highlevels of IL-11Rα expression in blood vessels. The skilled artisan willrealize that IL-11Rα staining may be of use to distinguishandrogen-dependent from androgen-independent cases and therefore toassist in tailoring therapeutic treatment to the status of the tumor asandrogen-dependent or androgen-independent.

It is concluded that expression of IL-11Rα is of use as a specificmarker for metastatic prostate cancer in bone tissue. The skilledartisan will realize that IL-11Rα staining may be used for detection,diagnosis and/or imaging of metastatic prostate cancer in bone and/orother tissues, such as lymph nodes.

Clinical Significance

Approximately half of presently hospitalized cancer patients will die oftheir disease despite optimal management. Given such a high failurerate, estimates of potentially curative treatment based on the risk ofrecurrence remain difficult to extrapolate for an individual cancerpatient. There is a clear need for improved biomarkers of cellulargrowth potential and targets in cancer. Based on the present results,expression of IL-11Rα in prostate cancer appears to be one suchbiomarker.

The molecular observations reported herein may be confirmed in aclinical context by following patient outcome in prostatic cancers withvarying levels of IL-11Rα expression, using known methods. For example,probabilities of survival for each group of patients may be analyzed bythe Kaplan-Meier method. Log-Rank test may be used to determinestatistical differences between groups. A Cox proportional hazards modelmay be used to analyze the effect of single and multiple risk factors inassociation with survival. Martingale residual plots may be used toassess the proportional hazard assumption. Results may be consideredstatistically significant at P <0.05.

Such observations may be validated in archived pathological material.Group stratification of cancer patients based on a panel of one or moresuch markers would allow less aggressive tumors to be effectivelyeradicated, while patients with more aggressive tumors could be offeredexperimental therapies earlier in their clinical progression. Withoutreliable ways of predicting which tumors will progress, many cases aretreated aggressively on the chance of cure, but often at the price ofpotentially devastating treatment-associated side effects. There is aclear need for markers of cellular growth potential, such as IL-11Rα, asdiagnostic and therapeutic targets in cancer patients. The skilledartisan will realize that expression of IL-11Rα may be useful in othertypes of tumors besides prostate cancer, so long as IL-11Rα iscorrelated with tumor growth and/or metastatic potential. Exemplarytumors in which IL-11Rα may be of use for detection, diagnosis and/orprognosis of cancer include prostate, breast, colon, ovary and melanoma.

Example 4 Biopanning Circulating Immunoglobulins in Human ProstateCancer Patients

A phage display library was screened against a pool of circulatingantibodies obtained from a human prostate cancer patient. The biopanningprocedure resulted in the identification of a novel marker for prostatecancer that is diagnostic for disease progression in metastatic prostatecancer. In this embodiment, the antibody pool provides a structuralsampling of ligands targeted to naturally occurring receptors, some ofwhich may constitute novel disease markers. Biopanning against anantibody pool may be used to identify disease markers and to furthercharacterize the molecular events underlying the disease state.

The present Example shows the feasibility of this approach byidentifying a novel marker for prostate cancer. The results further showthat this marker has prognostic value for predicting which individualswith prostate cancer are likely to have an unfavorable clinical outcome,resulting in death of the patient. As discussed above, there is a greatneed in the field of prostate cancer for a reliable method to separatethose individuals whose prostate cancer will prove lethal (and thereforeare candidates for more aggressive therapeutic intervention) fromindividuals who will not die from prostate cancer. The present Examplerepresents a significant advance in prostate cancer prognosis andillustrates the utility of the claimed methods and compositions.

The skilled artisan will realize that although the present Example dealswith prostate cancer, the methods and compositions disclosed aresuitable for use with any disease state or condition in which the hostimmune system is likely to produce antibodies against a molecular markerassociated with the disease or condition.

The repertoire of circulating antibodies from the serum of prostatecancer patients with advanced disease was used to screen a phage displaylibrary. Certain peptides binding to those antibodies correspond totumor antigens expressed in bone metastasis of prostate cancer. A panelof prostate cancer serum samples from patients with recorded clinicaloutcome was screened by an ELISA assay against those peptides. Theresults show that reactivity against one particular peptide (“peptideC”) can be used to identify patients with metastaticandrogen-independent prostate cancer. Moreover, patients with detectablelevels of circulating antibodies against peptide C exhibited decreasedsurvival compared to individuals without such antibodies.

Methods

Sera was selected from patients diagnosed with androgen-dependent andandrogen-independent prostate cancer. A CX6C peptide library wasscreened against this pool of IgGs in a two-step procedure. First, thepeptide library was pre-cleared against a pool of purified IgGs fromnormal serum samples using Protein G affinity chromatography. This stepremoved peptides from the phage display library that bound toimmunoglobulins from patients without prostate cancer. Next, thepre-cleared peptide library was screened against the pool of purifiedIgGs from the serum of prostate cancer patients. This step selectedpeptides binding specifically to IgGs elicited against prostate cancer.

Human Sera and Tissue Samples

Human plasma samples were prospectively collected from 91 patients withlocally advanced, metastatic androgen-dependent and metastaticandrogen-independent adenocarcinoma of the prostate. In each case, thepatient was evaluated in reference to tumor staging (locally advanced ormetastatic disease) and hormone responsiveness of the disease(androgen-dependent or androgen-independent). Criteria for enrollmentconsisted of a combination of the TNM classification and histologicalgrading. Patients diagnosed with adenocarcinoma of the prostate withstage T1c or T2 with Gleason score less than or equal to 7 and serum PSA<10 ng/ml were considered to have clinically organ-confined prostatecancer. Study entry in the locally advanced group required appropriateprimary tumor staging (stage T_(1c) or T₂ with Gleason score greaterthan 7; or clinical stage T_(2b-2c) with Gleason score equal to orgreater than 7 and serum PSA >10 ng/ml; or clinical stage T₃) and noregional (N₀) or distant (M₀) metastases. Study entry in the metastaticgroup required evidence of regional (N₁) and/or distant (M₁) metastasesin radionuclide bone scan, chest radiography, or computed tomography ofthe abdomen and pelvis. Androgen-independence was defined as serumtestosterone lower than 50 ng/dl and serially rising serum PSA; indexpatients 1, 2, and 4 were androgen-independent, while index patient 3was androgen-dependent at the time their serum samples were obtained.

For biopanning, sera was examined from three metastaticandrogen-independent and one metastatic androgen-dependent prostatecancer patients. Plasma from 34 healthy individual donors (eleven males)was obtained from the Blood Bank at the University of Texas M. D.Anderson Cancer Center (UTMDACC). Archived tissue paraffin blocks wereobtained from the Department of Pathology at UTMDACC. The blood sampleswere initially allowed to clot at room temperature and then centrifugedto separate the cellular component from the supernatant. Aliquots ofsupernatant were promptly frozen and stored at −80° C. until assayed.

Biopanning.

A 6-mer cyclic peptide (CX₆C) phage display library was used for thebiopanning. To select peptides specific to the serum antibodies ofcancer patients, a pre-clearing stage was employed to removenon-specific peptides by pre-absorbing the peptide library onto purifiedIgGs from pooled normal serum (five healthy male individuals). Thepre-cleared peptide library was screened onto the purified IgGs from theserum of prostate cancer patients. In brief, 10⁹ transducing units(T.U.) of a CX₆C cyclic peptide phage library were incubated with IgGantibodies from 50 μl of normal serum immobilized on 50 μA of protein G(Gibco BRL) for 1 hour at 4° C. This was followed by affinity selectionon the immobilized IgG antibodies from prostate cancer patient serum for2 hours at 4° C. Phage peptides specifically bound to IgGs elicitedagainst prostate cancer were eluted with 100 μl of 0.1 M glycine buffer,pH 2.2, neutralized by the addition of 10 μl 1M Tris pH 9.0, and used toinfect E. coli strain K91. Ten-fold serial dilutions of the infectedsolution were spread onto agar plates containing 40 μg/ml oftetracycline and grown overnight. Two hundred colonies were picked,amplified, and precipitated for a subsequent round of panning. A totalof three rounds were performed. Individual phage clones were picked forPCR and the insert DNA was sequenced.

Enzyme-Linked Immunosorbent Assay and Peptide Inhibition Study.

A 20 μg/ml solution of purified GST or GST-fusion proteins in 0.1MNaHCO₃ was used to coat maxisorp multi-well plates (Nalge NuncInternational Corporation) and incubated overnight at 4° C. The plateswere blocked in a blocking buffer (4% milk, 2% casein, and 0.05%Tween-20) for 3-4 hours. A series of 100-fold dilutions (1:100-1:1200)of sera from prostate cancer patients or healthy individuals was addedand incubated for 1.5 hours and then washed five times with washingbuffer (1% milk, 0.5% casein, and 0.025% Tween-20), followed byincubation at 4° C. with anti-human alkaline phosphatase-conjugatedantibodies (Gibco). The plates were then washed six times in washingbuffer and developed using p-NPP (Sigma) as a substrate. An automaticELISA plate reader (BIO-TEK instrument) recorded the results at OD405nm.

Antibody Biotinylation.

GST-fusion proteins containing the peptide sequence from patient C werecoated on multi-well plates. After incubating the plates with thepatient's serum, the plates were washed. The bound IgG antibodies wereeluted with 50 μl of 0.1 M glycine buffer, pH 2.2, neutralized byaddition of 10 μl 1 M Tris pH 9.0, and dialyzed in PBS overnightfollowed by concentration of the antibody using Centricon-30 (Millipore)filters. The purified antibody (500 μg) was coupled to biotin accordingto the manufacturer's instructions (Vector). The biotinylated antibodywas analyzed by SDS-gel electrophoresis.

Immunohistological Staining.

Paraffin sections (4 μm) were stained with purified biotinylatedantibodies and peptide antibodies by immunoperoxidase detection usingthe Dako antigen retrieval kit and DAB (diaminobenzidine) as asubstrate. All of the sections were counter-stained with hematoxylin.Purified IgGs were coupled to biotin and resolved by SDS-PAGE. Thebiotinylated immunopurified antibodies were used at a dilution of 1:60.Peptide C antibodies and purified pre-immune antibodies were used at0.01 μg/μl. For the inhibition staining, peptide C antibodies werepre-incubated for 30 minutes at room temperature with the correspondingGST-peptide C (500 μg) prior to staining. For the GRP78 immunostaining,anti-GRP78 antibody (C-20) was used at 1:350 (Santa Cruz Biotechnology,Santa Cruz, Calif.). Peptide antibodies were generated in rabbits andpurified using a T-gel immunoglobulin purification kit and protein Gcolumn (Pierce).

Protein Purification, Mass Spectrometry, and Immunoprecipitation

DU-145 prostate cancer cells (American Type Culture Collection), whichexpress the native antigen (data not shown), were used for proteinpurification. Cells were grown to 70% confluence, harvested in PBS, andtreated with TM buffer (100 mM Tris-Cl, 2 mM MgCl₂, 1% Triton-X100).Cells were sheared to separate nuclei from cytoplasm and otherorgandies. The cytosolic/membrane fraction was centrifuged. Thesupernatant was collected, resolved on 4-20% gradient SDS-PAGE, probedby rabbit anti-peptide antibodies on Western blots and detected byenhanced chemiluminescence (ECL; Pharmacia). The band containing theprotein recognized by the anti-serum was excised and used for proteinsequencing. Mass spectrometry analysis was compared to databasescontaining known protein sequences by BLAST homology search.

For immunoprecipitation, 200 μl of protein G agarose beads (Pierce) werecoupled to anti-GRP78 or rabbit anti-peptide antibodies, and therecombinant GRP78 (Stressgen) was added at 150 μg, and incubated for 4hours. As a negative control, protein G agarose beads alone were used.The immunoprecipitates were recovered by centrifugation, rinsed withwash buffer (0.05% Tween-20 in PBS), and resolved by SDS-PAGE.

A Western blot was probed with either anti-CNVSDKSC (SEQ ID NO:39) oranti-GRP78 antibodies (each at 1:200 dilution) and detected by ECL. Fordetection of GRP78 in the normal prostate and bone metastasis, wholelysates from frozen tissue samples were prepared by grinding the tissuein a dounce homogenizer in a 2 ml of Tissue Protein Extraction Reagent(Pierce) per sample with protease inhibitors (10 μg/ml of leupeptin andaprotonin). The homogenate was incubated on ice for 10 minutes prior torepeated grinding. The homogenate was spun at 610 g for 5 minutes andthe supernatant was removed and protein concentration was measured usingthe Protein DC Assay (BIO-RAD). 20 μg of protein from the normalprostate and bone metastasis lysates were resolved on a 4-20% SDS-PAGE,probed by anti-GRP78 antibody on Western blots and detected by ECL.

Cross-Inhibition Assays

Microtiter 96-well plates were coated with 10 μg/ml recombinant GRP78(Stressgen) or GST attached to CNVSDKSC (SEQ ID NO:39) in 100 mM NaHCO₃overnight at 4° C., washed and then blocked with blocking buffer (2%milk, 1% casein, 0.05% Tween-20 in PBS) for 2 hours at 37° C. Todetermine the inhibitory activities of GRP78 or GST-CNVSDKSC (SEQ IDNO:39), patient serum (1:50), anti-GRP78 (1:1000), and anti-GST-CNVSDKSC(1:20) were incubated with GRP78 (50-100 μg) or GST-CNVSDKSC (SEQ IDNO:39) (100-300 μg). The mixtures were incubated for 1 hour at 37° C.prior to adding to the coated wells. After 1 hour of incubation at roomtemperature the wells were washed several times with PBST buffer (0.05%Tween-20 in PBS). Secondary antibodies conjugated to horseradishperoxidase were added at 1:5000 dilution, incubated for 30 minutes atroom temperature and washed five times with PBST buffer. Finally, thesubstrate 3,3′,5,5′-Tetramethylbenzidine (TMB; Calbiochem) was added andincubated for 15 minutes at room temperature before stopping thereaction by addition of 0.5M H₂SO₄. Absorbance at 450 nm was determinedin an automated ELISA reader (Bio-Tek).

Statistical Analysis.

Probabilities of survival for each group were estimated using theKaplan-Meier method. A log-rank test was implemented in order to detectsignificant differences between the groups. Reactivity was considered tobe detected if the ratio between GST-peptide and GST alone was greateror equal to two by the ELISA data. A Log-Rank test was used to determinestatistical differences between groups. A statistical CART analysis(Classification And Regression Tree) was used to identify the bestcut-off point for determining reactivity to GRP78. In this method, thecensored survival data were transformed into a single uncensored datavalue (the so-called “null martingale residual”), which was used asinput into a standard regression tree algorithm. A cut-off point of 0.95was determined by this program

Results

After three rounds of selection, a striking enrichment (log scale) wasobserved in three out of the four serum samples examined (FIG. 4). Inthe fourth patient sample, no enrichment was observed and this patientwas not studied further. Individual phage clones from the second andthird rounds of selection from serum samples A, B, and C were sequenced.The peptide motifs CHQKPWEC (SEQ ID NO:40) from patient A and CKDRFERC(SEQ ID NO:41) from patient B represented 100% of the clones analyzedfrom those patients. In patient C, the peptide CNVSDKSC (SEQ ID NO:39)appeared in 55% of the clones analyzed. The remaining clones identifiedin patient C were CNWTDKTC (SEQ ID NO:43), representing 33.3% of theclones in round II and 7% of the clones in round III, CNITQKSC (SEQ IDNO:44), representing 33.3% of the clones in round II and 0% in roundIII, and CNKTDKGC (SEQ ID NO:45), representing 16.7% of the clones inround II and 0% in round III.

ELISA was performed to assess if the peptides could be specificallyrecognized by the antibodies present in the serum of the patientsselected for the screenings. Peptides A (CHQKPWEC, SEQ ID NO:40), B(CKDRFERC, SEQ ID NO:41), and C (CNVSDKSC, SEQ ID NO:39) were producedas GST-fusion proteins and immobilized onto microtiter wells, along withGST alone as a negative control. For each sample tested, a series of100-fold dilutions was performed. Little reactivity occurred with theGST control, whereas strong reactivity occurred with the GST-fusionpeptides (FIG. 5). The reactivity of each serum against peptides A, Band C was inhibited by the corresponding synthetic peptides (data notshown).

Characterization of the Peptide CNVSDKSC (SEQ ID NO:39) and ClinicalCorrelations

Having shown selective binding of GST-CNVSDKSC (SEQ ID NO:39) fusionpeptides to prostate cancer patient serum, the reactivity profileagainst the peptide CNVSDKSC (SEQ ID NO:39) was assessed in a populationof 108 sera obtained from clinically annotated prostate cancer patientsand 71 age-matched healthy men (negative control). Among the controlserum samples tested, a small percentage of positive reactivity (7%) wasdetected with the selected peptide (FIGS. 6A and 6B). In contrast,positive serum reactivity from the 108 sera samples from prostate cancerpatients correlated positively with the natural progression of thedisease (FIGS. 6A and 6B). Thus, positive serum reactivity againstCNVSDKSC (SEQ ID NO:39) correlated with late-stage prostate cancer andandrogen-independence. For example, only 6% of the organ-confinedpatients' sera reacted against the peptide CNVSDKSC (SEQ ID NO:39),whereas patients with androgen-dependent tumors reacted against thepeptide in 29% of the samples (FIG. 6B). Most notably, 76% of thesamples obtained from patients with metastatic androgen-independentprostate cancer reacted to the sequence CNVSDKSC (SEQ ID NO:39) (FIG.6B).

Kaplan-Meier curve estimates (Kaplan and Meier, J. Am. Statist. Assoc.53:457-481, 1958) were applied to compare survival between the positivereactive and negative reactive groups (FIG. 6C). Reactivity against thepeptide CNVSDKSC (SEQ ID NO:39) (n=42) was associated with asignificantly shorter patient survival (FIG. 6C, Log-Rank test, P=0.02).The median survival in the positive reactivity group was reached after32.7 months while the median survival in the non-reactivity group wasnot reached (FIG. 6C). The data show a strong correlation betweenpositive reactivity against the peptide CNVSDKSC (SEQ ID NO:39),development of metastatic androgen-independent prostate cancer (the mostadvanced stage of the disease), and decreased survival.

Identification of the Corresponding Native Tumor-Associated Antigen.

Antibodies against the peptide sequence CNVSDKSC (SEQ ID NO:39) wereused to determine whether they could specifically recognizetumor-associated targets in tissue sections by immunohistochemistry.Normal prostate tissue and metastatic prostate cancer from bone marrowin samples obtained from patient C were subject to IHC staining usingautologous immunopurified IgGs or a rabbit polyclonal antibody againstCNVSDKSC (SEQ ID NO:39). Strong staining was observed usingimmuno-purified antibodies from the autologous patient serum (FIG. 7A).Specific immunostaining was also observed using a rabbit polyclonalantibody raised against the synthetic peptide CNVSDKSC (SEQ ID NO:39)(FIG. 7B). No immunostaining was observed with the pre-immune antibodies(FIG. 7C) or a secondary antibody alone (FIG. 7D). Theimmunohistochemical signal observed in FIG. 7B was mostly inhibited by afusion protein containing the sequence CNVSDKSC (SEQ ID NO:39)demonstrating the specificity of the staining protocol (FIG. 7E). Normalprostate from the same individual only exhibited weak staining using theantibody against CNVSDKSC (SEQ ID NO:39) (FIG. 7F).

The target antigen mimicked by the peptide sequence CNVSDKSC (SEQ IDNO:39) was identified by standard biochemical techniques. An extract ofthe DU145 prostate cell line, containing cytosolic and cell membranefractions, was prepared as disclosed above and reacted withanti-CNVSDKSC (SEQ ID NO:39) polyclonal antibody, prepared by injectingrabbits with CNVSDKSC (SEQ ID NO:39) conjugated to KLH. A single 80 KDaprotein was identified by Western blotting (not shown).

The 80 kDa band was excised for protein sequencing. Five peptidesequences were obtained from the protein excised from SDS gels. All fivepeptides matched portions of the 78 kDa glucose regulated protein (Table6, GRP78, SEQ ID NO:42, GenBank Accession Numbers CAB71335 and XM044202). The locations of the five sequenced peptides within GRP78 areindicated in Table 7 in bold font. A commercial antibody against GRP78(Santa Cruz Biotechnology, Santa Cruz, Calif.) reacted on Westernblotting with the purified 80 kDa peptide C antigen from DU145 cells(not shown). The original peptide C sequence (SEQ ID NO:39) is not foundwithin the GRP78 sequence (SEQ ID NO:42), indicating that the epitoperecognized in vivo by anti-peptide C antibodies is formed fromdiscontiguous regions of the GRP78 protein.

TABLE 7 Sequence of Human GRP78 (SEQ ID NO: 42)     MKLSLVAAMLLLLSAARAEEEDKKEDVGTVVGIDLGTTYSCVGVFKNGRVEIIANDQGNRITPSYVAFTPEGERLIGDAAKNQLTSNPENTVFDAKRLIGRTWNDPSVQQDIKFLPFKVVEKKTKPYIQVDIGGGQTKTFAPEEISAMVLTKMKETAEAYLGKKVTHAVVTVPAYFNDAQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLDKREGEKNILVFDLGGGTFDVSLLTIDNGVFEVVATNGDTHLGGEDFDQRVMEHFIKLYKKKTGKDVRKDNRAVQKLRREVEKAKRALSSQHQARIEIESFYEGEDFSETLTRAKFEELNMDLFRSTMKPVQKVLEDSDLKKSDIDEIVLVGGSTRIPKIQQLVKEFFNGKEPSRGINPDEAVAYGAAVQAGVLSGDQDTGDLVLLDVCPLTLGIETVGGVMTKLIPRNTVVPTKKSQIFSTASDNQPTVTIKVYEGERPLTKDNHLLGTFDLTGIPPAPRGVPQIEVTFEIDVNGILRVTAEDKGTGNKNKITITNDQNRLTPEEIERMVNDAEKFAEEDKKLKERIDTRNELESYAYSLKNQIGDKEKLGGKLSSEDKETMEKAVEEKIEWLESHQDADIEDFKAKKKELEEIVQPIISK LYGSAGPPPTGEEDTAEKDEL

The molecular mimicry between the selected peptide and GRP78 was shownby reciprocal co-immunoprecipitation with either anti-GRP78 antibody oranti-peptide CNVSDKSC (SEQ ID NO:39) antibody (not shown). Whole lysateswere made from frozen tissue samples of normal prostate and bonemetastasis. Equivalent amounts of protein (20 ug) were resolved on 4-20%SDS-PAGE and probed with an anti-GRP78 antibody on Western blots. GRP78was weakly expressed in normal prostate tissue, whereas it was highlyexpressed in the bone metastasis from a patient with prostate cancer(not shown). Recombinant GRP78 or the GST-CNVSDKSC (SEQ ID NO:39) fusionprotein were capable of blocking binding to the 80 kDa protein of thepatient's serum antibodies, the anti-GRP78 antibody, and polyclonalantibodies raised against the peptide CNVSDKSC (SEQ ID NO:39) (FIG. 8).Collectively, these data demonstrate that GRP78 is the endogenousantigen against which circulating antibodies are present in a highpercentage of metastatic prostate cancer patients.

Prognosis and Predictive Value of Serum Reactivity to GRP78.

GRP78 functions in antigen presentation (Melnick & Argon, Immunol. Today16:243-50 1995). Its stress-responsive promoter is strongly induced inresponse to glucose deprivation, acidosis, and chronic hypoxia Lee,Trends Biochem. Sci. 26:504-510, 2001). Since such conditions aregenerally present in poorly vascularized solid tumors, it was determinedwhether GRP78 is a general biomarker of the tumor microenvironment orwhether its expression is specific to prostate cancer.

The reactivity of serum samples obtained from prostate cancer patientsand controls was evaluated against GRP78. Using a cut-off point of 0.95absorbance as determined by the “CART” (Classification And RegressionTree) statistical method, a 26-52% positive reactivity was observed in apopulation of patients with advanced prostate cancer in contrast to only6% in age-matched control men and 0% in the organ-confined group (FIG.9A). GRP78 reactivity was also examined in the serum of three groups ofnon-prostate cancer patients (FIG. 9A). Significantly less reactivityagainst GRP78 was observed in serum from patients with metastaticnon-small cell lung cancer (P<0:001), metastatic breast cancer (P<0.001)and advanced ovarian cancer (P<0.001) (FIG. 9A).

A survival curve was applied to compare the overall survival between thepositive reactivity and non-reactivity groups (n=108) for GRP78 (FIG.9B). Positive reactivity to GRP78 was associated with a shorter survivaloutcome (Log-Rank test, P=0.07) (FIG. 9B, lower line). Taken together,these data strongly suggest that reactivity against GRP78 is apreferential serological marker of prostate cancer relative to othermalignant tumors.

Expression of GRP78 in Bone Metastasis and Normal Prostate Tissue.

The presence of circulating antibodies against GRP78 was associated withthe most aggressive stage of prostate cancer (metastaticandrogen-independent disease). The expression of GRP78 was examined byimmunohistochemical analysis in normal prostate tissue and bone marrowmetastasis from a prostate cancer. The GRP78 antigen was highlyexpressed in bone marrow metastasis as shown by strong immunostaining(FIG. 10A), whereas weak staining was observed in normal prostate tissue(FIG. 10B). These results confirm the Western analysis using the sametissue samples noted above (FIG. 7F). To show specificity, staining wasinhibited using recombinant GRP78 (FIG. 10C) or the peptide fusionprotein GST-CNVSDKSC (SEQ ID NO:39) (FIG. 10D). These data demonstratethat GRP78 is highly expressed in prostate cancer metastases to bonemarrow and weakly expressed in normal prostate tissue.

Discussion

The present Example shows that it is possible to identify molecularmarkers of disease progression and survival without prior knowledge ofthe antigens related to the disease. In cases where the tumor antigen isunknown, disease-specific antigens identified by this approach could beemployed to define common or unique features in the immune response ofindividuals to the same disease, i.e., to fingerprint the immuneresponse against a given antigen. The approach presented here is basedon selection of immunoglobulin-binding peptides that mimic tumor-relatedantigens from phage libraries. Serum samples from human prostate cancerwere screened and an antibody-binding peptide ligand was validated byusing a large panel of patient serum samples. The corresponding tumorantigen eliciting the immune response was identified as GRP78, amolecular marker of use for detection, diagnosis and/or prognosis ofmetastatic prostate cancer. The GRP78 protein is highly expressed inbone marrow metastasis and the high prevalence of circulating antibodiesagainst GRP78 is associated with metastatic androgen-independent diseaseand poor prognosis.

GRP78 (also known as Hsp70 protein 5) expression is induced by cellularstress and hypoxia, conditions associated with prostate cancer.Recently, this protein has been shown to be abundant in malignantprostate tumor by two-dimensional electrophoresis and mass spectrometry(Alaiya et al., Cell Mol. Life. Sci. 58:307-11, 2001). In addition toGRP78, other heat shock proteins, such as 90, 72, and 27, are highlyexpressed in malignant prostate tissue (Thomas et al., Br. J. Urol.77:367-72, 1996). GRP78 associates with the major histocompatibilitycomplex (MHC) class I on the cell surface and its presence on the cellsurface is not dependent on MHC class I expression (Triantafilou et al.,Hum. Immunol. 62:764-70, 2001). Cancer-derived HSP-peptide complexes arebeing used as HSP vaccine in human cancer (Tamura et al., Science278:117-120, 1997). A recent study showed that the expression of heatshock proteins could independently determine the clinical outcome ofindividual prostate cancers (Tamura et al., 1997).

Although phage peptide libraries have been used to identify variouspathological and disease-related agents in patients including Lymedisease, hepatitis, HIV-1, and autoimmune diseases, this is the firstreport in which sera from prostate cancer patients have been used toidentify new markers for this cancer.

Example 5 Biopanning Circulating Antibodies in Prostate Cancer AntibodyProgression Corresponds to Disease Progression

The present Example illustrates a further embodiment of the invention,using phage display library screening to examine the progression incirculating antibodies accompanying disease progression in prostatecancer.

The methods used were similar to those described in Example 4. Asubtraction protocol was used, in which IgG from a normal individual wascoupled to protein G chromatography beads. A cyclic CX₆C phage displaylibrary, prepared as described above, was exposed to the normal IgG's.Phage that did not bind to the normal IgG pool were collected and usedfor the next step. Antibodies from patient M (prostate cancer patient)were attached to fresh protein G chromatograpy beads. The phage displaylibrary that had been pre-exposed to normal IgG's was exposed to the IgGpool from patient M. After thorough washing of the column, the phagethat bound to the prostate cancer IgG (but did not bind to normal IgG)was eluted and amplified. This procedure was followed for three roundsof screening and targeting peptides against patient M's antibodies wereobtained.

Serum samples from the same patient were obtained from archivalspecimens and used to obtain targeting peptides. Patient M's serum from1994 (early stage cancer), 1998 (intermediate stage) and 2000 (latestage) were used to obtain antibody targeting peptides as describedabove. These peptides were shown in Table 8. The numbers in parenthesesindicate the number of phage exhibiting the sequence out of the totalnumber of clones obtained.

TABLE 8 Peptides identified after three rounds of pan- ning on purified immunoglobulins from the serum  of prostate cancer patient M. 1994 Serum 1998 Serum 2000 SerumCTFAGSSC (6/22) CTFAGSSC (12/20) CTFAGSSC (26/29) (SEQ ID NO: 46)(SEQ ID NO: 46) (SEQ ID NO: 46) CNSAFAGC (1/22) CSKKFVTC (3/20)CNSAFAGC (1/29) (SEQ ID NO: 47) (SEQ ID NO: 62) (SEQ ID NO: 47)CSYTFAGC (1/22) CNSAFAGC (1/20) CFPKRVTC (1/29) (SEQ ID NO: 48)(SEQ ID NO: 47) (SEQ ID NO: 66) CSTFAGSC (1/22) CKNKHTTC (1/20)CPRSAKNC (1/29) (SEQ ID NO: 49) (SEQ ID NO: 63) (SEQ ID NO: 67)CRDGYHHC (1/22) CFETFAGC (1/20) (SEQ ID NO: 50) (SEQ ID NO: 64)CSASDLSC (2/22) CNNMYAGC (1/20) (SEQ ID NO: 51) (SEQ ID NO: 65)CQNQYPEC (1/22) CQNQYPEC (1/20) (SEQ ID NO: 52) (SEQ ID NO: 52)CRASAMVC (1/22) (SEQ ID NO: 53) CIDMTHQC (1/22) (SEQ ID NO: 54)CISSPSNC (1/22) (SEQ ID NO: 55) CNQSMWSC (1/22) (SEQ ID NO: 56)CQFENGTC (1/22) (SEQ ID NO: 57) CAVKSVTC (1/22) (SEQ ID NO: 58)CNGFMGYC (1/22) (SEQ ID NO: 59) CLTSENAC (1/22) (SEQ ID NO: 60)CRASAMVC (1/22) (SEQ ID NO: 61)

It is apparent that one sequence, CTFAGSSC (SEQ ID NO:46) was thepredominant antibody-binding peptide in all three samples. Further, thefrequency of this targeting peptide as a fraction of the total pool oftargeting peptides increased with time, suggesting that the antibodythat bound this peptide also became more prevalent with tumorprogression. It is also apparent that the diversity of targetingpeptides binding to circulating antibodies decreased with diseaseprogression, indicating that there was a corresponding decrease inantibody diversity.

It is not unusual for tumor cells to shed antigens into the circulation.Leukocytes may also be exposed to tumor antigens in situ. It istherefore expected that cancer patients in general will exhibitcirculating antibodies against tumor antigens. Phage display librariesmay be screened against cancer patient samples to identify targetingpeptides that bind to antibodies against tumor specific or tumorassociated antigens. The identified targeting peptides may be used, forexample, to purify anti-tumor antibodies using affinity chromatograpy orother well-known techniques. The purified anti-tumor antibodies can beused in diagnostic kits to identify individuals with cancer.Alternatively, they could be attached to various therapeutic moieties,such as chemotherapeutic agents, radioisotopes, anti-angiogenic agentsor pro-apoptosis agents and used for cancer therapy. The targetingpeptides against anti-tumor antibodies may also be used to identifynovel tumor specific or tumor-associated antigens, of diagnostic ortherapeutic use. Phage display antibody libraries may also beconstructed and screened against tumor targeting peptides. By thismethod, it is possible to isolate and purify large quantities ofantibodies specific for tumor antigens.

The skilled artisan will realize that the CTFAGSSC (SEQ ID NO:46)peptide could be used for ELISA or other immunoassays to screen theblood of individuals at risk for prostate cancer. The presence of anantibody that bound to SEQ ID NO:46 in the serum of a patient would beindicative of prostate cancer. The peptide may also be used to preparemonoclonal or polyclonal antibodies that are of use for tumor diagnosis,imaging or therapy.

Example 6 Targeted Phage-Based Vectors for Systemic Gene Delivery

Certain embodiments of the present invention concern gene therapyvectors for treatment of various cell, tissue or organ-localized diseasestates, such as prostate cancer. Targeting peptides may be incorporatedinto or attached to therapeutic vectors and administered to patientswith the disease, decreasing the systemic toxicity of the therapeuticagent and increasing its targeting to the diseased tissue, therebyincreasing efficacy. In particular embodiments, the gene therapy vectorsof use include, but are not limited to, modified adeno-associated virus(AAV) vectors, referred to herein as adeno-associated phage (AAP)vectors. The AAP vector enables systemic and local gene delivery androbust long-term transgene expression. The vector specifically homes toreceptors that have been well characterized for selective expression onthe vascular endothelium. The AAP vector can deliver genes to angiogenicor tissue-specific receptors. It results in markedly increasedtransduction stability and duration of gene expression

The development of vectors for systemic targeted delivery is requiredfor successful gene therapy. Commonly used approaches rely on ablatingthe native tropism of viral vectors and/or retargeting them toalternative receptors. Thus far, a major drawback of these approacheshas been that the expression of the receptors is not restricted to thetarget tissues.

Many malignant, cardiovascular, and inflammatory diseases have a markedangiogenic component. In cancer, tumor vasculature is a suitable targetfor intervention because the vascular endothelium is composed ofnon-malignant cells that are genetically stable but epigeneticallydiverse (St. Croix, B. et al., Science 289:1197-1202, 2000; Kolonin etal., Curr. Opin. Chem. Biol. 5:308-313, 2001). In vivo phage display hasbeen used to isolate probes that home selectively to different vascularbeds and target receptors expressed only on certain blood vessels. Bothtissue-specific and angiogenesis-related vascular ligand-receptor pairshave been identified with this technology. Targeted delivery ofcytotoxic drugs (Arap et al., Science 279:377-380, 1998a), proapoptoticpeptides (Ellerby et al. Nat. Med. 5:1032-1038, 1999), fluorophores(Hong & Clayman, Cancer Res. 60:6551-6556, 2000) or cytokines (Curnis etal., Nat. Biotechnol. 18:1185-1190, 2000) to the vasculature generallyimproved selectivity and/or therapeutic windows in animal models.Vascular receptors are attractive targets for systemic delivery of genetherapy. Such receptors are readily accessible through the circulationand often can mediate internalization of ligands by cells (Kolonin etal., 2001).

While incorporation of vascular homing peptides derived from in vivophage display screenings into viral vectors has been attempted, thisstrategy has proven quite challenging because the structure of thecapsid and the targeting properties of the peptides can be adverselyaffected (Wickham, Gene Ther. 7:110-114, 2000). However, gene expressionin mammalian cells is possible if phage vectors are processed in thecorrect trafficking pathway (Poul & Marks, J. Mol. Biol. 288:203-211,1999).

In theory, phage vectors have several advantages over mammalian virusesconventionally used for gene therapy. Receptors for prokaryotic virusessuch as untargeted (wild-type) phage are not expressed on mammaliancells. Receptor-mediated internalization by mammalian cells does occurif re-targeted phage vectors display certain peptide ligands (Larocca etal., Faseb J. 13:727-734, 1999). There is substantial evidencesuggesting that phage can be safely administered to patients, asbacteriophage were given to humans during the pre-antibiotic era with noadverse effects (Barrow & Soothill, Trends Microbiol. 5:268-271, 1997).Because homing phage have been pre-selected to home to vascularreceptors in an in vivo screening, there is no need for furthertargeting modifications. The localization of gene expression in vivorecapitulates previous observations using immunohistochemistry for phagelocalization (Rajotte et al., 1998; Rajotte & Ruoslahti, 1999;Pasqualini et al., 1997). The parental tumor-homing phage used in thepresent Example are known to target receptors expressed in the activatedblood vessels of multiple types of human and murine tumors, includingcarcinomas, melanomas, and sarcomas in mouse models (Pasqualini et al.,1997; Arap et al., 1998; Koivunen et al., 1999a). The lung-homing phageand its corresponding receptor expressed in the lung vasculature havealso been well characterized in mice (Rajotte et al., 1998; Rajotte &Ruoslahti, 1999).

Based on the rationale outlined above, targeted systemic gene deliveryto the vascular endothelium may be accomplished with phage particleshoming to cell surface receptors on blood vessels while meeting receptorrequirements for selective tissue expression and vector accessibility.The results presented herein demonstrate the feasibility of thisapproach.

A new generation of targeted phage-based vectors is provided thatenables systemic gene delivery and robust long-teem transgeneexpression. A novel chimeric phage-based vector containing the invertedterminal repeat (ITR) sequences from adeno-associated virus (AAV) hasbeen designed, constructed, and evaluated. As demonstrated below, thesevectors (i) specifically home to receptors that have been wellcharacterized for selective expression on the vascular endothelium, (ii)can deliver genes to angiogenic or tissue-specific blood vessels, and(iii) markedly increase transduction stability and duration of geneexpression. These data indicate that targeted phage-based vectors andtheir derivatives are of use for clinical applications, such as targeteddelivery to prostate cancer.

Materials and Methods

Reagents, Cells, and Tissue Culture

All of the restriction enzymes (New England Biolabs, Beverly, Mass.), T4DNA ligase (Roche, Indianapolis, Ind.), topotecan (Sigma ChemicalCompany, St. Louis, Mo.), and cisplatin (Sigma) were obtainedcommercially. The fMCS1 plasmid was obtained from Dr. George P. Smith(University of Missouri, Mo.). DNA sequence analysis was performed withthe Big Dye® terminator sequence kit (Perkin Elmer/ABI Systems, Norwalk,Conn.). All peptides used in this Example were synthesized at greaterthan 95% purity, cyclized, and analyzed by HPLC and mass spectrometry(AnaSpec, San Jose, Calif.). Targeting peptides used in this Exampleincluded the GFE (CGFECVRQCPERC, SEQ ID NO:68); HWGF (CTTHWGFTLC, SEQ IDNO:69) and RGD-4C (CDCRGDCFC, SEQ ID NO:70) peptides.

The human cell lines used were Kaposi's sarcoma (KS1767), 293 embryonickidney (ATCC; Manassas, Va.), and MDA-MB-435 breast carcinoma. Celllines were maintained in minimal essential medium (MEM; IrvineScientific, Santa Ana, Calif.) supplemented with 10% fetal calf serum(FCS; Gibco-BRL, Rockville, Md.) plus sodium pyruvate, L-glutamine, andpenicillin/streptomycin (Gibco-BRL).

Construction of Phage-Based Targeted Expression Vectors

The FUSES-based filamentous phage display vector was modified byinserting into an intergenic region of the phage genome aβ-galactosidase (β-gal) coding sequence under the control of a CMVpromoter, Targeted RGD4C-β-gal phage vector was engineered in a two-stepprocess that included the generation of an intermediate construct(termed RGD-4C-fMCS1) and subsequent production of RGD-4C-β-gal. Theoverall construction scheme is illustrated in FIG. 11. RGD-4C-fMCS1contained the oligonucleotide insert encoding the RGD-4C targetingpeptide, inserted into the Sfi I site of the gene III minor coat proteinof the FUSES phage, and a fragment of the fMCS1 plasmid that had amulticloning site (MCS) for insertion of transgenes. RGD-4Cphage-derived fUSE5 DNA and fd-tet phage-derived fMCS1 DNA were purifiedfrom lysates of host bacteria (E. coli MC1061). The intermediateRGD-4C-fMCS1 vector was constructed by ligating a 5.4-kb BamHI/SacIIfragment of the RGD-4C plasmid to the 4.1 kb BamHI/SacII fragment of thefMCS1 plasmid. Next, a 14 kb RGD-4C-β-gal phage plasmid was obtained byinsertion of a 4.5 kb PstI CMV-β-gal fragment derived from pCMVβ(Clontech, Palo Alto, Calif.) into the PstI site of RGD-4C-fMCS1. Thisstrategy allowed cloning of the CMV-β-gal cassette in either forward orreverse orientation.

Orientations of resulting vectors were differentiated by EcoRVrestriction analysis and by DNA sequencing. Targeted phage vectors weredesignated fRGD4C-β-gal (forward) and rRGD4C-β-gal (reverse). Othertargeting (HWGF-β-gal, GFE-β-gal) phage and insertless control(fd-β-gal) phage were constructed through the same strategy. Thetargeting phage were designed to target integrins (RGD-4C) and the MMP-2and MMP-9 matrix metalloproteinases (HWGF), expressed in angiogenicvasculature. The GFE phage were designed to target membrane dipeptidase(MDP) expressed in lung vasculature.

A targeted phage/AAV chimeric vector was created by cloning a 2.8 kbfragment of pAAV-eGFP (enhanced GFP; Stratagene) from ITR (invertedterminal repeat) to ITR into the PstI site of RGD-fMSC. Briefly, pAAVwas digested with Pad to release a 2.8 kb fragment, which was bluntedwith DNA polymerase and cloned into the blunted PstI site of RGD-fMSC(thus destroying the PstI restriction site). The final AAP vectorconstruct is illustrated in FIG. 32. The 12.3 kb DNA contains atargeting motif inserted into gene III, a gene of interest (e.g., β-gal)inserted between the AAV ITR elements under control of a CMV promotorand with a poly A terminator. The locations of the deleted Pst I sitesare also shown (crossed out). In each of the constructs, correctorientation of insert was verified by restriction analysis. Singleclones in each orientation were sequenced. Unless otherwise stated, theforward vectors were used.

Phage DNA Transfection into Mammalian Cells

The double-stranded DNAs of the replicative forms of targeted(RGD4C-β-gal, HWGF-β-gal, GFE-β-gal) and insertless control (fd-β-gal)constructs were prepared by using the Plasmid Maxi kit (Qiagen). Thesingle-stranded DNAs of the infective forms of the phage vectors wereextracted from the phage capsid proteins by using the Strataclean resin(Stratagene), followed by two ethanol precipitations. DNA was quantifiedby spectrophotometry with 1.0 A₂₆₀ equal to 40 μg/ml for single-strandedDNA or 50 μg/ml for double-stranded DNA. The 293 recipient cells weretransfected with 5 μg of either double-stranded or single-stranded phageDNA into 5×10⁵ cells by using the SuperFect® reagent (Qiagen) accordingto the manufacture's protocol. Both the gene expression and enzymeactivity of β-gal were evaluated at least 48 hours post-transfection.Cells were incubated with the X-gal substrate for 3 hours at 37° C. andenzyme activity was visualized by using an in situ β-galactosidasestaining kit (Stratagene) according to the manufacturer's instructions.

Vector Production, Purification, and Titration.

Phage vectors were isolated and purified from the culture supernatant asdisclosed (Pasqualini et al., in Phage Display: A Laboratory Manual(Barbas et al., eds.), chap. 22, pp. 1-24, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2000). Phage were re-suspended inTris-buffered saline (pH 7.4) and re-centrifuged to remove residualbacteria and debris. The resulting supernatant containing the phage insuspension was filtered through a 0.45 μm filter and titered accordingto standard protocols (Pasqualini et al., 2000).

Targeted phage vector transduction and specific inhibition by usingsynthetic peptides. MDA-MB-435 breast cancer and KS1767 Karposi'ssarcoma cells were cultured on 8-well chamber glass slides. The culturemedia was replaced by 200 μl of MEM with 2% FCS and 5×10¹⁰ TU ofRGD-4C-β-gal, HWGF-β-gal, or fd-β-gal phage vectors (at 10⁵ transducingunits/cell in each case). Both cell lines express high levels of theintegrin and MMP receptors for those targeting peptides. Phage wereincubated with cells for 3 hr at 37° C., followed by a medium change toMEM plus 10% FCS. The cells were incubated for 72 hr at 37° C. to allowfor n-gal gene expression.

In the peptide inhibition studies, MDA-MB-435 cells were cultured on12-well plates and then incubated with 10 μg of RGD-4C peptide orcontrol peptides (CARAC, SEQ ID NO:71 or CKDRFERC, SEQ ID NO:41) innormal growth media for 30 minutes. KS1767 cells were grown on 12-wellplates and then incubated with 40 μg CTTHWGFTLC (SEQ ID NO:69) orcontrol peptides in normal growth media for 30 minutes. The growth mediawere replaced by 500 μl of MEM containing 2% FCS and 5×10¹⁰ transducingunits (TU) of either RGD-4C-β-gal, HWGF-β-gal, or control fd-β-galphage. Phage vectors were incubated on peptide-treated cells (threehours at 37° C., 5% CO₂) followed by a media change to MEM plus 10% FCS.Transduced cells were maintained in a cell incubator for 72 hours (37°C., 5% CO₂).

In the cell culture transduction assay, β-gal expression was analyzed byimmunofluorescence. For quantification of expression in cell culture,the transduced cells were washed with PBS and permeabilized with 0.2%Triton X-100 for five minutes on ice, followed by blocking with 1% BSAin PBS. An anti-β-gal antibody (Sigma) diluted to 1:2,000 in blockingsolution was then incubated with the cells overnight. Next, a TexasRed-conjugated secondary antibody (Caltag, Burlingame, Calif.) dilutedto 1:600 in PBS was incubated with the cells for 1 hour. The degree ofβ-gal gene expression was determined by counting fluorescent cells in atleast ten fields under an inverted microscope (Nikon, Japan).Quantification of the β-gal activity in cell culture was measured asrelative light units (RLU) in a luminometer and then normalized to theamount of protein in micrograms, as determined by the Lowry method in aprotein assay kit (Bio-Rad Protein Assay®; Hercules, Calif.).Subsequently, blue cells were counted under an inverted microscope(Nikon).

In the peptide inhibition assays, β-gal activity in cell lysates wasdetected by the Galacto-Star® chemiluminescent reporter gene system(Tropix, Bedford, Mass.) according to the manufacturer's protocol. Inother peptide inhibition assays, 293 cells were plated at 3×10⁵cells/well and incubated with either 1 mg/ml of RGD-4C peptide orunrelated control peptides (CARAC, SEQ ID NO:71 or CKDRFERC, SEQ IDNO:41). After 30 minutes, cells were washed and 10⁵ TU of phage per cellwere added for 4 hours in serum free media. After the 4 hours, 10% FCSsupplemented medium was added. Cells were analyzed for GFP geneexpression at 72 hours post infection. For GFP detection, cells wereanalyzed by fluorescence activated cell sorting (FACS) in a FACScan(Becton-Dickinson, San Jose, Calif.) or counted and photographed under afluorescence microscope (Nikon).

For time course of gene expression assays, cells were plated at 3×10⁵cells/well and infected with 10⁵ TU of phage per cell for 4 hours inserum-free media. After 4 hours, 10% FCS supplemented medium was added.Cells were visualized 72 hours post-infection and sorted by FACS for GFPexpression 7 days after infection. GFP-positive cells were plated in T75tissue culture flasks and serial assays of GFP expression as describedabove were made weekly for the next 60 days.

Genotoxic Agents.

Semi-confluent MDA-MB-435 cells were infected with 10⁵ TU of phage percell for 4 hours in serum free media, after which fresh mediumsupplemented with FCS was added (no phage were washed out or removed).In some experiments, a phage admixture of forward and reverse clones at10¹⁰ TU (forward/reverse molar ratio=1) was tested. Next, cells wereincubated for 36 hours followed by the addition of genotoxic drugs(topotecan, 10 μM; cisplatin, 10 μM) or administration of UV radiation(15 J/m²) with a cross-linker apparatus (UV Stratalinker Model 2400;Stratagene). At 72 hours post-infection, the cells were analyzed fortransduction of a reporter gene (β-gal or GFP), and gene expression wasnormalized per cell number relative to controls.

In Vivo Transduction of Tumor Xenografts and Normal Lung in MouseModels.

Female 4-month old nude mice and female 4-month old immunocompetentC57B1/6 mice (Harlan Sprague Dawley, San Diego, Calif.) were used inthis study. Avertin (0.015 ml/g) was used as an anesthetic. Tumorxenografts derived from human Kaposi's sarcoma KS1767 cells wereestablished by injecting tumor cells (10⁶ cells per mouse in 200 μl ofserum-free MEM) into the mammary fat pad of nude mice. Tumor-bearingmice with matched tumor sizes were used for systemic gene transferexperiments 20 to 40 days afterwards when tumors reached 0.5 to 1.5 cmin diameter.

In tumor transduction experiments, RGD-4C-β-gal, HWGF-β-gal, andfd-β-gal phage (10⁹ TU/mouse) were injected intravenously (tail vein)into female nude mice carrying subcutaneous tumor xenografts. One weekafter vector administration of the targeted or control phage, tumors andcontrol organs (liver, brain) were surgically harvested under deepanesthesia and the mice euthanized. β-gal expression in the tumor andcontrol tissues was detected by an anti-β-gal antibody by using aperoxidase-based immunodetection kit (Vector Labs, Burlingame, Calif.).

In lung transduction studies, GFE-β-gal phage and fd-β-gal control phage(10⁹ TU/mouse) were injected intravenously into female C57B1/6 mice.Lungs and livers were harvested two weeks after vector administration.For in vivo experiments involving tissue extracts, β-gal activity in thelung and control tissues were detected by a chemiluminescent assaysystem (Tropix). Several assays for β-galactosidase were used indifferent studies to ensure that the results were not assay-dependentand were reproduced with distinct methods.

AAP Vector for Delivery of Therapeutic Genes to Tumors

The efficacy of the AAP vector to deliver therapeutic genes to Karposi'ssarcoma tumors in nude mice was evaluated. The most frequently usedsystem of gene delivery consists of transferring the Herpes simplexvirus type 1 (HSV-1) thymidine kinase (TK) gene into tumor cells,followed by treatment with Ganciclovir (GCV). This guanosine analogue isspecifically monophosphorilated by the viral kinase and then convertedby cellular enzymes into the triphosphate derivative, which, uponincorporation into elongating DNA, induce cell death, by premature chaintermination. To determine whether AAP vector could be used for systemicgene therapy delivery, the βl cassette was replaced with a “suicide”gene (thymidine kinase—TK). The resulting RGD-4C-AAP-TK vector wasinjected intravenously in nude mice bearing human KS1767 Kaposi'ssarcoma xenografts. Targeting, internalization and transduction of thetherapeutic AAP vector into the tumor cells, followed by treatment withGCV should result in cell death.

Molecular Characterization of AAP Vectors

Viral rescue experiments were performed in AAP-transduced 293 cells byinfecting them with Ad5 (MOI of 10 particles/cell). After 48 hours thecells were processed to obtain a crude viral lysate, then heatinactivated to remove contaminating adenovirus. The resulting materialwas next used to infect 293 cells and 8431 cells. GFP-expressing cellswere detected after 48 hours. PCR analysis was performed by analysis ofgenomic DNA extracted from AAP-transduced 293 cells and from controlcells. Genomic DNA (200 μg/reaction) was reacted with GFP specificprimers (GFP-N, by 143-164; GFP-C, by 654-676). After 30 PCR cycles, thepresence of a diagnostic 490 bp band is evaluated. To ensure specificamplification, pCMV-GFP DNA was used as a positive control and pCMV DNAwas used as a negative control. Southern Blot analysis was performedwith Eco RI-digested genomic DNA extracted from AAP-transduced 293 cellsand controls. The digests were electrophoresed and hybridized with a³²P-labeled cDNA fragment containing an AAV-specific probe. The presenceof a diagnostic 2.3 kb band was evaluated.

Results

Targeted Phage Vectors Designed to Drive Gene Expression in EukaryoticCells.

The fUSE5-based filamentous phage display vector (Smith & Scott, 1993)was modified by inserting the β-galactosidase (β-gal)-encoding geneunder the control of a CMV promoter into an intergenomic region of thephage genome to construct a fUSE5-β-gal backbone vector. Next, DNAolignonucleotide sequences encoding the targeting peptides CDCRGDCFC(SEQ ID NO:70, “RGD-4C”), CTTHWGFTLC (SEQ ID NO:69, “HWGF”) orCGFECVRQCPERC (SEQ ID NO:68, “GFE”) were inserted into the Sfi I site ofthe gene III minor coat protein (pIII) of the phage. Phage produced inthis manner display 3-5 copies of the targeting peptides per viralparticle.

The resulting viral constructs (RGD-4C-β-gal, HWGF-β-gal, and GFE-β-gal)were used for production of targeted phage particles that display eachof the targeting peptides and carry a CMV-β-gal transgene (FIG. 11).RGD-4C-β-gal and HWGF-P-gal were designed to target αv integrins andmatrix metalloproteinases (MMP-2 and MMP-9), respectively, expressed inangiogenic vasculature. GFE-β-gal was designed to target membranedipeptidase (MDP) expressed in lung vasculature. The strategy depictedin FIG. 11 was used to construct the other targeting and controlvectors.

Phage DNA Context Permits Transgene Expression in Mammalian Cells

To determine whether the inserted β-gal cassette was functional,embryonic human kidney cells were transfected with the infective formsof the phage DNA, constructed to contain the reporter transgene ineither forward or reverse orientation. A CMV-driven mammalian expressionvector was used as a positive control (FIG. 12A) and an empty vector asa negative control (FIG. 12B) for β-gal expression. Transfer of themodified single-stranded DNA of the phage infective form promotedtransgene expression in mammalian cells. Furthermore, the orientation ofthe transgene cassette did not significantly influence the level of geneexpression (FIG. 12C vs. FIG. 12D). All subsequent experiments used thevector with the β-gal expression cassette in the forward orientation.Given that single-stranded DNA does not support gene expression inmammalian cells and that the infective forms of the phage genome aresingle-stranded, these results strongly suggest that the single-strandedphage genome must be first converted to double-stranded DNA in recipientcells before allowing gene expression.

Consistent with this hypothesis, DNA from replicative forms of thephage, which are double-stranded, expressed the β-gal transgene severalfold more efficiently at levels comparable to the mammalian expressionvector used as the positive control (data not shown).

Receptor-Mediated Internalization And Specific Transduction of RecipientCells by Targeted Phage Vectors In Vitro,

Having shown that the transgene constructs were functional, transductionof human cell lines expressing the receptors targeted by RGD-4Cβ-gal andHWGF-β-gal phage vectors was examined. The untargeted fUSE5-derivedcontrol phage vector (fd-β-gal) was used as a negative control.RGD-4C-β-gal phage (FIG. 13C-D) and HWGF-β-gal phage (FIG. 13A-B) wereincubated with breast cancer (FIG. 13C-D) and Kaposi's sarcoma (FIG.13A-B) cells (MDA-MB-435 and KS1767 lines), respectively. Both celllines express high levels of the RGD-4C-receptors αvβ3 and αvβ5integrins and of the HWGF receptors MMP-2 and MMP-9.

β-gal transduction was observed of 14±2% (mean±standard error of themean; SEM) of MDA-MB-435 cells incubated with RGD-4C-β-gal phage and12±2% (mean±SEM) of the KS1767 cells incubated with HGWF-β-gal (FIG.14A). Comparable transduction results were also obtained by incubatingHWGF-β-gal on MDA-MB-435 cells and RGD-4C-β-gal on KS1767 cells (datanot shown). Control phage (fd-β-gal) were not internalized whenincubated with either cell line and only minimal β-gal transduction(˜0.1% of the tumor cells) could be detected (FIG. 14A).

To demonstrate specificity, transduction with RGD-4C-β-gal andHWGF-β-gal phage was blocked by pre-incubating the target cells with thecorresponding synthetic peptides (FIG. 14B-C). In each case, almostcomplete inhibition of transduction was observed, of greater than 99%with RGD-4C peptide (FIG. 14C) and greater than 90% with CTTHWGFTLC (SEQID NO:69) peptide (FIG. 14B) in a dose-dependent manner. Pre-incubationwith nonspecific negative control peptides had no significant effects ontransduction of the recipient cells (FIG. 14 B-C). These data show thattransduction of mammalian cells by internalized phage vectors in vitrois substantial, specific, and mediated by ligand-receptor mechanisms.

Targeted Transduction of Tissue-Specific and Tumor Vasculature UponSystemic Administration In Vivo

To determine whether the targeted RGD-4Cβ-gal and HWGF-β-gal phagevectors could selectively transduce tumors upon systemic administration,each vector was administered intravenously into nude mice bearing humanKS1767 Kaposi's sarcoma xenografts. KS1767 cells are suitable becausethey form well-vascularized tumors and the receptor expression profilesin tumor cells and tumor-associated blood vessels has been characterized(Pasqualini et al., 1997; Arap et al., 1998a, 1998b; Koivunen et al.,1999a). The αv integrins and gelatinases (MMP-2 and -9) receptors forthe targeting peptides are highly expressed on the KS1767-derived tumorxenografts and their angiogenic vasculature. Phage displaying RGD-4C andHWGF peptides target KS1767 tumors efficiently and specifically in vivo.Tumor targeted phage were not detected in control tissues studied,including brain, kidney, pancreas, adrenal, skin, muscle, intestine,lymph nodes, uterus, prostate, and fat (Pasqualini et al., 1997; Arap etal., 1998a, 1998b; Koivunen et cd., 1999a).

Tumors and control organs were surgically harvested one week afteradministration of the vectors. Tumor and control organs (liver andbrain) were immunostained with an anti-β-gal antibody. The RGD-4C-β-gal(FIG. 15A, D and G), HWGF-β-gal (FIG. 15B, E, and H), and controlfd-β-gal (FIG. 15C, F, and I) vectors were analyzed.

Strong β-gal immunostaining was observed in tumor tissues (FIGS. 15A andB), with negligible immunostaining observed in control liver and brainorgans (FIG. 15D, E, G and H). In contrast, tissues recovered from micethat received untargeted negative control fd-β-gal phage vector did notshow detectable β-gal expression in either the tumor (FIG. 15C) or thecontrol organs (FIGS. 15F and I). In each case, β-gal reactivity matchedthe corresponding immunostaining pattern of phage targeting to thevascular endothelium of blood vessels in tumors (Pasqualini et al.,1997; Arap et al., 1998; Koivunen et al., 1999). A non-β-gal-containingphage produced no staining in the liver (data not shown). Measuringβ-gal activity produced results consistent with the immunohistochemistrydata used for detection of targeted gene transduction (FIG. 16).

Targeted gene delivery was also evaluated in vivo by using GFE-β-gal, aphage vector targeted to MDP in the vascular endothelium of lung bloodvessels (Rajotte & Ruoslahti, 1998). The lung-homing GFE-β-gal vectorwas injected intravenously into immunocompetent C57B1/6 mice.Substantial β-gal activity was seen in the lungs of mice injected withGFE-β-gal phage but not in the lungs of mice injected with fd-β-galcontrol (FIG. 17). In contrast, the β-gal activity in the liver of miceinjected with the GFE-β-gal phage was similar to that of backgroundβ-gal activity from mice injected with control phage (FIG. 17). Takentogether, these results show in vivo systemic gene delivery andtransduction targeted to and mediated by vascular receptors selectivelyexpressed in tumors and in normal organs.

Increase in Transduction by Genetic Trans-Complementation.

Because the genome from the infective form of M13-derived phage issingle-stranded, conversion to double-stranded DNA is required to allowgene expression. It was hypothesized that genotoxic agents that promoteDNA repair would enhance the transduction of genes carried bysingle-stranded phage vectors. To test this hypothesis, cells infectedby targeted phage vectors were challenged with genotoxic agents such asultraviolet (UV) radiation and cancer chemotherapy drugs (topotecan andcisplatin). This approach consistently resulted in gene transductionseveral fold higher than various controls (FIG. 18). Interestingly, anequal mixture of forward and reverse single-stranded phage clones showeda two-fold increase in gene expression relative to the same molarconcentrations of either forward or reverse phage (FIG. 18). It ispostulated that the presence of both sense and anti-sense of thereporter gene allowed hybridization of the strands to occur. Suchfacilitation in gene expression is consistent with the requirement fordouble-stranded DNA. The enhancement of gene expression by DNA lesionsor genetic trans-complementation indicates that conversion todouble-stranded DNA is a rate-limiting step in developing of effectivephage vectors. These data also suggest the possibility of synergism ifcytotoxic agents commonly utilized in clinical applications are used incombination with phage-derived vectors.

Phage/AAV Chimeric Vectors Markedly Improve Gene Transduction Stability.

To solve the DNA conversion problem described above, it was determinedwhether the incorporation of genetic cis-elements derived from AAV (asingle-stranded mammalian virus) into targeted phage-based constructswould affect gene transduction. First, chimeric vectors composed of atargeted phage and an AAV genome from inverted terminal repeat (ITR) toITR was designed and engineered. Vectors were constructed by cloning afull-length 2.8 kb fragment of pAAV-eGFP (Green Fluorescent Protein,Stratagene) from inverted terminal repeat (ITR) to ITR into the bluntedPstI site of the construct presented in FIG. 11. The targetingproperties of the resulting chimeric vectors were not altered byinsertion of AAV genetic elements. Specific inhibition by thecorresponding synthetic peptide was again observed (FIG. 20) indicatingthat the phage targeting features were intact.

Having established that the tropism of the targeted vector waspreserved, the effects of AAV genome insertion on transgene expressionwas assessed. While the levels of gene expression remained unchanged(data not shown), the duration of gene transduction was markedlyprolonged relative to the parental targeted phage. Robust long-termexpression of the reporter gene was observed beyond eight weeks (FIG.21). This finding is in clear contrast to the one-week transgeneexpression usually observed with the parental targeted phage vector invitro (Table 9). Table 9 represents relative reporter gene expression in293 cells transfected with targeted phage versus AAP, using triplicatewells for each time point. Results represent averaged independentdeterminations by two different investigators. Expression in thestandard targeted phage vector disappeared after 10 days, while someexpression was observed in AAP vectors for at least 60 days.

TABLE 9 Transgene expression in vitro Post-infection day # 0 5 0 0 5 0 50 5 0 Targeted phage + AAP + + + + + + + +

Tumor targeted transgene expression was also observed in vivo aftersystemic AAP administration in mice bearing MDA-MB-435 xenografts. Suchtransduction was specific because it was blocked by co-administration ofcognate—but not unrelated control—synthetic peptides (not shown).

The combination of genotoxic agents plus insertion of AAV cis-elementsappears to be at least additive if not synergistic (data not shown).Gene expression in cells transduced with targeted phage/AAV chimericvectors has been systematically followed for up to 60 days (Table 8).Expression of GFP has been detected for as long as 90 days (not shown).To rule out the possibility of genetic complementation by trans-actingfactors (for example, E4 or f6) in the permissive 293 cell line, thetransduction of HepG2 (liver carcinoma-derived) and MDA-MB-435 cells wasexamined. Similar levels and duration of gene expression were observed(data not shown).

To characterize the AAP chimeric vectors, studies were performed todetect AAV elements in cells transduced with AAP vectors and todemonstrate excision, amplification, and integration (FIG. 22).Adenoviral rescue (FIG. 22), PCR (not shown) and Southern Blot analysis(not shown) demonstrate that (i) AAV particles can be generated usingthe supernatant from cells infected with AAP; and (ii) AAV elementsintegrate within the genome in cells transduced with targeted AAP, butnot control phage vectors (targeted or untargeted).

These data indicate that phage/AAV chimeric (AAP) vectors may be readilyconstructed and used with no apparent losses in their targeted acquiredtropism and with substantial enhancement in the long-term stability ofthe genes transduced.

Delivery of Therapeutic Genes Into Tumors

An AAP vector designed to contain a “suicide” TK gene was constructed asdescribed above and injected into nude mice containing Karposi's sarcomahuman tumors. Seven (7) days after vector injection, mice received dailyintraperitoneal injections of GCV (5 mg/Kg/day) for 7 days. Tumors inanimals injected with RGD-4C-TK-AAP vector, followed by GCV treatment,showed significant growth reduction, comparing to tumors in the controlanimals which were injected with insertless fd-AAP-TK vector prior toGCV treatment. These results demonstrate the feasibility of using AAPvectors for targeted delivery of therapeutic genes to tumors and othertissues for which selective and/or specific targeting peptide sequenceshave been identified. The skilled artisan will realize that the AAPvector described herein is not limited to targeted delivery to tumortissues, but may be used for targeted gene therapy of a wide variety oforgans, tissues or cell types.

Discussion

The present Example shows for the first tune that systemic gene deliverycan be achieved by genetically adapting targeted phage clones selectedfrom screenings of phage display random peptide libraries. Thecharacteristics of an efficient phage-based gene therapy vector include:[1] selectivity towards target tissues; [2] receptor-mediated cellinternalization; and [3] long-term duration of gene transduction upondelivery. Each of these characteristics was exhibited by the AAP vectorsdisclosed herein.

Targeting peptides can be integrated into conventional gene therapyvectors or even used as bi-functional molecular adaptors (Larocca etal., 1999; Wickham, 2000; Grifman et al., Mol. Ther. 6:964-975, 2001;Trepel et al., Hum. Gene Ther. 11:1971-81, 2000). These strategies haveproven to be technically challenging and not necessarily efficient.Issues of specificity and efficiency have been addressed by takingadvantage of peptide ligands selected from phage libraries in vitro andin vivo. The targeting phage obtained in screenings performed in vivoare often selected using a 3-minute circulation timeframe. Thus, it isunlikely that the phage exits the circulation. The selection strategy isdesigned to favor vascular targeting and the isolation of phage thattarget markers that are accessible to circulating ligands (i.e.,expressed in cells forming vascular endothelium).

Given that selection has already occurred for those particular clones,it is likely that such phage would meet criteria for tissue targeting.Here, it was demonstrated that the null-tropism of wild-type phagetowards mammalian cells can be modified to target and deliver genes toreceptors expressed on the vascular endothelium of normal organs (suchas the lung) and tumors. Thus, the phage vectors introduced by thisstudy have a number of potential advantages. Their targeting toselective vascular beds is based on receptor expression patterns thatare known and characterized. The receptors are accessible to circulatingprobes. These ligand-receptor pairs provide internalization of thevector into targeted cells.

While it has been shown that phage can promote gene expression in vitro,gene transduction in vivo after systemic administration of a targetedphage vector has not as yet been reported. A major limitation in thepractical use of phage vectors has been poor levels of transductionachieved in vivo. A possible cause of this is the low efficiency ofconversion from single-stranded to double-stranded DNA occurring inmammalian cells. To solve this problem two independent strategies wereapplied: (i) enhancement of gene transduction by genotoxic agents(cytotoxic drugs and UV radiation) which cause strand breaks and promoteDNA repair; and (ii) genetic incorporation of AAV cis-elements intotargeted phage vectors. The strategies are not mutually exclusive andmay be used together to further improve the efficiency of gene therapyin vivo.

The term adeno-associated phage (AAP) is used for the new class ofvectors for gene delivery described here. The biological features of AAPare distinct from either targeted phage or AAV. While the enhancedduration of gene transduction by AAP is similar to the long-termexpression patterns associated with AAV transduction, thereceptor-mediated targeting is characteristic of phage clones selectedin screenings. Thus, AAP are endowed with several advantages as a genetherapy vector. AAP are easy to produce in high titers in host bacteria.No helper viruses or trans-acting factors are needed. The native tropismof AAV for human cells is eliminated because there is no AAV capsidformation. The AAP vectors are presumed targeted because theyincorporate peptides that have been isolated in vivo and are defined bytheir ability to home to selective vascular beds. Gene transductionstability was compared between a targeted phage and AAP vectors.Targeted gene delivery specific to the ligand-receptor pair to which thephage is directed is possible, and gene expression is maintained forover two months (possibly because of DNA integration).

The results reported herein demonstrate that the targeting propertiesare preserved in the hybrid AAP (a feature conferred by the phage) andthat gene expression elicited by such vectors is robust (a featureconferred by the AAV elements). Data with the AAP in vivo appear toconfirm these contentions (not shown).

In summary, genetically modified phage have potential to be adapted astargeted gene delivery vectors to mammalian cells after systemicadministration. Based on the favorable targeting properties andlong-term duration of gene transduction of AAP, these vectors are of useas superior gene delivery tools.

The skilled artisan will realize that the AAP vectors are not limited tothe targeting peptides used in the present Example, but rather may takeadvantage of any of the targeting peptides known in the art or disclosedherein, such as the prostate cancer targeting peptides described above.Such AAP gene therapy vectors, designed to contained cytostatic,cytotoxic, pro-apoptotic, anti-angiogenic or other therapeutic genes maybe selectively and/or specifically targeted to tissues, such as cancertissues, prostate cancer tissues, and/or metastatic prostate cancertissues to provide a high efficacy of tumor treatment, while exhibitinglittle or no systemic toxicity.

Example 7 Identification of Mouse Adipose Targeting Peptides

The present Example concerns compositions and uses of novel adiposetargeting peptides and receptors. In certain embodiments, the peptidesand receptor targets may be of use for targeted delivery of therapeuticagents to tumors and/or normal adipose tissues.

Adipose Targeting Peptides

A substractive phage display protocol (see Example 8 below) was used toisolate fat targeting peptides from a genetically obese mouse (Zhang etal., Nature, 372:425-432, 1994; Pelleymounter et al., Science269:540-543, 1994). Phage that had been subjected to biopanning in obesemice were post-cleared in a normal mouse. The fat-targeting peptidesisolated included TRNTGNI (SEQ ID NO:72), FDGQDRS (SEQ ID NO:73); WGPKRL(SEQ ID NO:74); WGESRL (SEQ ID NO:75); VMGSVTG (SEQ ID NO:76), KGGRAKD(SEQ ID NO:77), RGEVLWS (SEQ ID NO:78), TREVHRS (SEQ ID NO:79) andHGQGVRP (SEQ ID NO:80).

Homology searches identified several candidate proteins as theendogenous analogs of the fat targeting peptides, including stem cellgrowth factor (SCGF) (KGGRAKD, SEQ ID NO:77), attractin (mahogany)(RGEVLWS, SEQ ID NO:78), angiopoietin-related adipose factor (FIAF)(TREVHRS, SEQ ID NO:79), adipophilin (ADRP) (VMGSVTG, SEQ ID NO:76),Flt-1 or procollagen type XVII (TRNTGNI, SEQ ID NO:72) and fibrillin 2or transferrin-like protein p97 (HGQGVRP, SEQ ID NO:80)

Validation of Adipose Targeting Peptides

The fat homing peptides were validated by in vivo homing, as shown inFIG. 23. The fat homing clones selected were: FA-KGGRAKD (SEQ ID NO:77),FC-RGEVLWS (SEQ ID NO:78), FE-TREVHRS (SEQ ID NO:79) and FX-VMGSVTG (SEQID NO:76). As seen in FIG. 23, all of these clones exhibited someelevation of homing to adipose tissue, with clone FX showing severalorders of magnitude higher adipose localization than control fd-tetphage. Clone FX also exhibited substantially higher localization thanthe other selected fat homing clones. However, by analogy with theplacental homing peptides disclosed above, the skilled artisan willrealize that fat homing clones exhibiting lower levels of adipose tissuelocalization may still be of use for targeted delivery of therapeuticagents.

The skilled artisan will realize that targeting peptides selective forangiogenic vasculature in adipose tissue could be of use for weightreduction or for preventing weight gain. By attaching anti-angiogenic ortoxic moieties to an adipose targeting peptide, the blood vesselssupplying new fat tissue could be selectively inhibited, preventing thegrowth of new deposits of fat and potentially killing existing fatdeposits.

Example 8 CKGGRAKDC (SEQ ID NO:81) Homes to White Fat in ob/ob Mice

Materials and Methods

Experimental Animals

C57BL/6 mice were purchased from Harlan Teklad. Leptin-deficient (ob/ob)(stock 000632) and leptin receptor-deficient (stock 000642) mice werepurchased from Jackson Laboratories (Bar Harbor, Me.). Anesthesia wasperformed with Avertin (0.015 ml/g) administered intraperitoneally(Arap, et al., 1998; Pasqualini & Rouslahti, 1996).

In Vivo Phage Library Screening

In vivo phage-display screening of the CX₇C library (C, cysteine; X, anyamino acid) (Pasqualini et al., 2000; Arap et al., Nature Med.8:121-127, 2002) for fat-homing peptides was performed (Pasqualini &Rouslahti 1996, Pasqualini et al., 2000). In each biopanning round, anadult ob/ob mouse was injected intravenously (tail vein) with 10¹°transducing units (TU) of the library. Phage (˜300 TU/g in round 1increased to ˜10⁴ TU/g in round 3) were recovered after 5 min ofcirculation by grinding subcutaneous white fat with a glass Douncehomogenizer, suspending the homogenate in 4° C. Dulbecco's ModifiedEagle's medium (DMEM) containing proteinase inhibitors (DMEM-prin: 1 mMPMSF, 20 μg/ml aprotinin, and 1 μg/ml leupeptin) and washing withDMEM-prin. The lipid phase was discarded during the washes and only thesolid-phase cellular material was used. Washed homogenates wereincubated with host bacteria (log phase E. coli K91kan; OD₆₀₀ ˜2).Bacterial cultures were plated onto Luria-Bertani agar plates containing40 μg/ml tetracycline and 100 μg/ml kanamycin, incubated overnight at37° C. and selected clones were bulk-amplified and used to precipitatephage for a subsequent round of biopanning. The sub-library amplifiedafter the third round of panning was enriched for fat-specific bindersusing a subtraction step. A lean C57BL/6 female was injected (tail vein)with 10⁹ TU of phage selected in round 3. After 5 min of circulation,the unbound phage were recovered from plasma and amplified for thefourth and final round of biopanning. In this protocol, phage that boundto tissues other than adipose were removed from the sub-library,increasing the selectivity of the recovered phage for binding to adiposetissue.

Peptide Localization in Tissues

Staining of formalin-fixed, paraffin-embedded mouse tissue sections wasperformed (Pasqualini & Rouslahti, 1996; Pasqualini et al., 2000). Forphage-peptide immunolocalization, 10¹⁰ TU of CKGGRAKDC (SEQ IDNO:81)-phage or a control insertless phage was injected intravenously.Phage immunohistochemistry was performed using a rabbit anti-fd phageantibody (Sigma Chemicals, St. Louis, Mo.) used at 1:1,000 dilution anda secondary horseradish peroxidase (HRP)-conjugated antibody. Apoptosiswas detected using standard TUNEL immunohistochemistry and anHRP-conjugated antibody. For in vivo peptide homing validation, stocksof 5-carboxyfluorescein (FITC)-conjugated CKGGRAKDC (SEQ ID NO:81) orCARAC (SEQ ID NO:71) were chemically synthesized, cyclized using theterminal cysteines and HPLC-purified to >90% purity by Anaspec (SanJose, Calif.). Lyophilized peptides were dissolved in DMSO to aconcentration of 20 mM. Ten μl of 1 mM peptide-FITC solution in PBS wasinjected 5 min prior to tissue extraction. For blood vessellocalization, 10 μl of 2 mg/ml of rhodamine-conjugated lectin-I(RL-1102, Vector Laboratories, Burlingame, Calif.) was co-injected. Allimmunohistochemistry and FITC immunofluorescence images were capturedusing an Olympus IX70 microscope and digital camera setup (Melville,N.Y.).

Anti-Obesity Therapy

Stocks of CKGGRAKDC (SEQ ID NO:81) fused to (KLAKLAK)₂ (SEQ ID NO:1);(KLAKLAK)₂ (SEQ ID NO:1) alone; CARAC (SEQ ID NO:71) fused to (KLAKLAK)₂(SEQ ID NO:1); and CKGGRAKDC (SEQ ID NO:81) peptide were chemicallysynthesized, cyclized using the terminal cysteines and HPLC-purifiedto >90% (Anaspec). Lyophilized peptides were dissolved in DMSO to aconcentration of 65 mM to make stock solutions. A total of 150 μl of0.65 mM peptide solution in PBS was subcutaneously injected daily in theback of C57BL/6 males, after body mass was measured each day. High-fatcafeteria diet for obesity induction (TD97366: 25.4% fat, 21.79%protein, 38.41% carbohydrate) was purchased from Harlan Teklad. Micewere pre-fed with TD97366 prior to the initiation of treatment withadipose targeting peptides to induce diet-related obesity. The high-fatdiet resulted in an average weight of 50 g before treatment.

Results

In vivo phage display (Pasqualini and Ruoslahti,. Nature 380:364-366,1996; Kolonin et al., Curr. Opin. Chem. Biol. 5:308-313, 2001;Pasqualini et al., In Vivo Phage Display, In Phage Display: A LaboratoryManual, eds. Barbas et al., pp. 1-24. Cold Spring Harbor LaboratoryPress, New York, 2000) was used as described above to obtain a peptidetargeting the fat vasculature. A phage-display library was screened forpeptide motifs that home to the vasculature of subcutaneous white fat inmorbidly obese leptin-deficient (ob/ob) mice (Zhang et al. Nature372:425-432, 1994). This model provides a convenient source of adiposetissue. Four rounds of panning were followed by a fat-specific in vivosubtraction to restrict ligands to those binding to adipose-specificendothelial receptors. The DNA encoding the correspondingphage-displayed peptides was then sequenced to obtain the targetingpeptide amino acid sequences. Statistical analysis of selected motifsusing SAS software (version 8, SAS Institute) revealed that the motifCKGGRAKDC (SEQ ID NO:81) constituted 4.5% of all clones identified inthe screen. Intravenous administration of this clone into ob/ob miceshowed that CKGGRAKDC (SEQ ID NO:81)-phage accumulated in subcutaneousfat to a higher level than a control insertless phage (data not shown).

The tropism of CKGGRAKDC (SEQ ID NO:81)-phage for adipose tissue wasconfirmed by immunohistochemistry: CKGGRAKDC (SEQ ID NO:81)-phage showedmarked localization to the vasculature of subcutaneous and peritonealwhite fat (FIG. 24 a, arrows), whereas the control phage wasundetectable in fat blood vessels (FIG. 24 b). To test whether targetingof the CKGGRAKDC (SEQ ID NO:81) motif to the fat vasculature would alsooccur when the peptide is outside of the context of the phage, the invivo distribution of intravenously injected CKGGRAKDC (SEQ ID NO:81)peptide fused to fluorescent (FITC) was determined. Immunofluorescencein subcutaneous and peritoneal fat from peptide-injected ob/ob miceshowed that CKGGRAKDC (SEQ ID NO:81)-FITC localized to and wasinternalized by cells of white adipose vasculature (FIG. 24 c, arrows),whereas a control CARAC (SEQ ID NO:71)-FITC conjugate was undetectablein adipose tissue (FIG. 24 d).

CKGGRAKDC (SEQ ID NO: 81) homes to white fat in wild-type mice

The mutation in leptin that leads to the extreme proliferation of whiteadipose tissue in mice (Zhang et al., 1994) is not frequentlyencountered in humans (Ozata et al., J. Clin. Endocrinol. Metab.84:3686-3695. 1999). Thus, this animal model may not be representativeof the typical pattern of obesity in humans. To exclude the possibilitythat CKGGRAKDC (SEQ ID NO:81) homing to fat is limited to ob/ob mice andto demonstrate the general applicability of adipose-targeting peptidesfor naturally-occurring obesity, the CKGGRAKDC (SEQ ID NO:81) peptidewas tested in wild-type mice.

FIG. 25 shows that the CKGGRAKDC (SEQ ID NO:81)—FITC fusion peptideintravenously injected into C57BL/6 (leptin +/+) mice specificallylocalized to blood vessels of subcutaneous and peritoneal white fat(FIG. 25A, FIG. 25B). A lectin-rhodamine peptide was used to visualizeblood vessel endothelium (arrows, FIG. 25B, FIG. 25D, FIG. 25F). TheCKGGRAKDC (SEQ ID NO:81)-FITC fusion peptide co-localized withlectin-rhodamine in adipose tissue (arrows, FIG. 25A and FIG. 25B). Nosuch co-localization was observed in control pancreatic tissue (FIG. 25Cand FIG. 25D) or other control organs (data not shown). The controlCARAC (SEQ ID NO:71)-FITC peptide was not detectable in white fatvasculature (FIG. 25E and FIG. 25F). These in vivo localization datashow that the adipose-targeting CKGGRAKDC (SEQ ID NO:81) peptide targetsthe white adipose vasculature in genetically normal obese mice as wellas in leptin deficient mice, demonstrating the general applicability ofadipose targeting using such peptides. The uptake of CKGGRAKDC (SEQ IDNO:81)-FITC by the endothelium of fat tissue suggests that the motiftargets a receptor selectively expressed in the adipose vasculature thatcould provide a mechanism for directed delivery of therapeutic compoundsto fat.

Design and Use of Fat-Targeted Pro-Apoptotic Peptide

It was next determined whether proliferation of adipose tissue could becontrolled via targeted destruction of the fat vasculature. Thepro-apoptotic peptide KLAKLAKKLAKLAK (SEQ ID NO:1) (Ellerby et al.,Nature Med. 5:1032-38, 1999), designated (KLAKLAK)₂ (SEQ ID NO:1), whichdisrupts mitochondrial membranes to induce apoptosis, has been targetedto receptors in tumor vasculature via a conjugated homing peptide(Ellerby et al 1999, Arap, et al., Proc. Natl. Acad. Sci. U.S.A.99:1527-1531, 2002). The (KLAKLAK)₂ (SEQ ID NO:1) peptide was conjugatedto the fat targeting CKGGRAKDC (SEQ ID NO:81) peptide for targeteddelivery to fat vasculature in adipose tissue. The D enantiomer of(KLAKLAK)₂ (SEQ ID NO:1), which is resistant to proteolysis but stillexhibits pro-apoptotic activity, was conjugated to the CKGGRAKDC (SEQ IDNO:81) peptide via a glycinylglycine bridge. The conjugatedfat-targeting, pro-apoptotic peptide was administered to mice and theeffect on adipose tissue was monitored.

A non-genetic mouse obesity model was initially used. A cohort ofC57BL/6 (wild-type) mice, in which obesity had been induced by ahigh-fat cafeteria diet, were subcutaneously injected with CKGGRAKDC(SEQ ID NO:81)-(KLAKLAK)₂ (SEQ ID NO:1) peptide and weighed daily over aperiod of two weeks. Cafeteria dieting continued throughout theexperiment. As shown in FIG. 26A, injections of CKGGRAKDC (SEQ ID NO:81)conjugated to (KLAKLAK)₂ (SEQ ID NO:1) prevented obesity development andsurprisingly caused a rapid decrease in body mass of up to 20%. Incontrast, obese mice injected with two negative controls (an equimolaramount of either unconjugated CKGGRAKDC (SEQ ID NO:81) and(KLAKLAK)_(2 (SEQ ID NO:)1) or a control CARAC (SEQ ID NO:71)-(KLAKLAK)₂(SEQ ID NO:1) conjugate) did not show a significant body mass decreaseand continued to increase in weight (FIG. 26A).

The effectiveness of the CKGGRAKDC (SEQ ID NO:81)-(KLAKLAK)₂ (SEQ IDNO:1) conjugate was also examined in wild-type mice fed on a regulardiet (FIG. 26B). C57BL/6 mice that had developed a considerable amountof subcutaneous and peritoneal fat due to old age were subcutaneouslyinjected with the CKGGRAKDC (SEQ ID NO:81)-(KLAKLAK)₂ (SEQ ID NO:1)conjugate or control peptides over a period of one month. As in thediet-induced obesity model, targeting of (KLAKLAK)₂ (SEQ ID NO:1) to fatby conjugation with CKGGRAKDC (SEQ ID NO:81) resulted in greater than35% reduction in body mass at a rate of 10% per week (FIG. 10B). Notoxicity of the conjugated peptide was detected under these conditions(data not shown). In fact, the CKGGRAKDC (SEQ ID NO:81)-(KLAKLAK)₂ (SEQID NO:1) treated mice became more active and agile following body massreduction and appeared healthier than prior to treatment (data notshown). The control untargeted (KLAKLAK)₂ (SEQ ID NO:1) treatmentsresulted in only a slight body mass reduction (FIG. 26B), possibly dueto low levels of nonspecific toxicity. The control mice did not exhibitthe increased activity and/or agility seen in treated mice (data notshown).

Fat resorption with CKGGRAKDC (SEQ ID NO: 81)- (KLAKLAK)₂ (SEQ ID NO: 1)is mediated by apoptosis

In both diet-induced and age-related obesity, the effect of CKGGRAKDC(SEQ ID NO:81)-(KLAKLAK)₂ (SEQ ID NO:1) treatment on body mass was dueto fat resorption, which was visually apparent by the end of treatment(FIG. 27). Wild-type mice were fed on a high fat cafeteria diet (FIG.27A). Alternatively, wild-type fed on a regular diet became obese as aconsequence of old age (FIG. 27B, FIG. 27C, FIG. 27D). Mice were treatedwith CKGGRAKDC (SEQ ID NO:81) conjugated to (KLAKLAK)₂ (SEQ ID NO:1)(left side of FIG. 27), with CARAC (SEQ ID NO:71) conjugated to(KLAKLAK)₂ (SEQ ID NO:1) (middle of figure), or with unconjugatedCKGGRAKDC (SEQ ID NO:81) and (KLAKLAK)₂ (right side of FIG. 27).

Gross inspection of mouse organs revealed that both subcutaneous (FIG.27B) and visceral (FIG. 27C) fat exhibited marked resorption upontreatment with CKGGRAKDC (SEQ ID NO:81) conjugated to (KLAKLAK)₂ (SEQ IDNO:1) (right side of FIG. 27). Quantification of fat resorption afterthree weeks of treatment by weighing a specific fat depot (epididymalfat, FIG. 27D) showed a greater than 3-fold reduction in fat masscompared with controls (FIG. 27D, left side of figure compared to middleand right side).

Histopathological analysis of tissues from mice treated with CKGGRAKDC(SEQ ID NO:81) conjugated to (KLAKLAK)₂ (SEQ ID NO:1) showed vascularapoptosis (FIG. 28A, arrows) and resulting fat necrosis with lymphocyteinfiltration (FIG. 28C, arrows) in adipose tissue. following treatment.In contrast, mice treated with a control fusion peptide comprising CARAC(SEQ ID NO:71) conjugated to (KLAKLAK)₂ (SEQ ID NO:1) showed no vascularapoptosis or fat necrosis (FIG. 28D). No abnormalities in other organstreated with CKGGRAKDC (SEQ ID NO:81) conjugated to (KLAKLAK)₂ (SEQ IDNO:1) (data not shown).

Injection of CKGGRAKDC (SEQ ID NO:81) conjugated to (KLAKLAK)₂ (SEQ IDNO:1) into genetically obese mice, but not into normal obese mice, wasoccasionally observed to result in mortality within a few days ofinjection. It is not clear what the mechanism might be for inducingdeath in genetically obese mice, although development of pulmonary orcardiac fat embolism or rapid drop of serum calcium due tosaponification by released lipids are possibilities. However, theseresults suggest that treatment of grossly obese subjects might result insufficient adipose cell death and necrosis to adversely affect thehealth of the subject, indicating that lower dosages and/or use of atime release formulation of the adipose targeting conjugate may bepreferred in cases of excessive obesity.

(SEQ ID NO: 81) Adipose receptor protein for CKGGRAKDC

A band of approximately 35,000 Daltons (35 kDa) was isolated from mouseadipose tissue extract that bound to CKGGRAKCDC (SEQ ID NO:81)conjugated to (KLAKLAK)₂ (SEQ ID NO:1) (not shown). There was much lessbinding of the 35 kDa fraction to the control peptide CARAC (SEQ IDNO:71) conjugated to (KLAKAK)₂ (SEQ ID NO:1) (data not shown). The 35kDa band was analyzed by mass spectrometry, which identified threeproteins present in the sample.

The three proteins included predominately a B cell receptor associatedprotein (prohibitin), apolipoprotein E, and the voltage dependent anionchannel (VDAC). Further studies were performed by immunoprecipitation,using either CKGGRAKDC (SEQ ID NO:81) or CARAC (SEQ ID NO:71) conjugatedto (KLAKAK)₂ (SEQ ID NO:1) and precipitating with commercially availableantibodies.

SDS-polyacrylamide gel electrophoresis of the immunoprecipitated proteinshowed that only the prohibitin receptor protein complex wassubstantially enriched by binding to CKGGRAKDC (SEQ ID NO:81) (data notshown), with over a ten-fold enrichment in the CKGGRAKDC (SEQ ID NO:81)precipitated fraction compared to the CARAC (SEQ ID NO:71) precipitatedfraction (data not shown). The CARAC (SEQ ID NO:71)-(KLAKAK)₂ (SEQ IDNO:1) fusion peptide exhibited low levels of non-specific binding to allthree proteins (VDAC, prohibitin and apolipoprotein E). It is unknownwhether those proteins bound to the CARAC (SEQ ID NO:71) moiety or to(KLAKAK)₂ (SEQ ID NO:1).

It is concluded that the adipose tissue endothelial receptor forCKGGRAKDC (SEQ ID NO:81) is prohibitin (Genbank Accession No.NM_(—)008831). Probitin is expressed in mitochrondria of various celltypes and in the cell membrane of B lymphocytes, where it is associatedwith the IgM receptor (McClung et al., Exp. Gerontol. 30:99-124, 1995).Based on these results, it is concluded that pro-apoptosis agentsconjugated to targeting peptides that bind to a prohibitin receptorprotein complex may be effective to induce adipose cell death and weightloss in obese subjects. The skilled artisan will realize that otherprohibitin-binding targeting peptides, antibodies, etc. may be usedwithin the scope of the claimed methods and compositions to controlweight and/or to induce weight loss. Further, other known cytocidal,cytotoxic and/or cytostatic agents may be used in place of (KLAKAK)₂(SEQ ID NO:1) to control weight or induce weight loss within the scopeof the claimed subject matter.

The results obtained in a mouse model system were confirmed in humantissue sections. Rabbit polyclonal antibodies against prohibitin werecommercially purchased (RDI-PROHIBIT, Research Diagnostics, Inc.,Flanders, N.J.). Immunohistochemistry on sections of fixed humanparaffin-embedded tissues was performed using the LSAB+peroxidase kitfrom Dako (Carpinteria, Calif.). Comparison of prohibitin expression inmouse versus human white fat tissue showed that prohibitin is highlyexpressed in blood vessels of both mouse and human white fat tissues(not shown).

Prohibitin is expressed in the vascular endothelium of a number of humanorgans (FIG. 30, arrows), including white fat tissue (FIG. 30A), skin(FIG. 30B), prostate (FIG. 30C) and bone (FIG. 30E). However, the levelof prohibitin expression in white fat blood vessels is much higher thanin other types of human tissues (FIG. 30).

Prohibitin expression appears to be inversely correlated with the degreeof malignancy in human adipose tissues (FIG. 29). The arrows indicateprohibitin staining in normal human white fat tissue (FIG. 29A), normalhuman breast tissue (FIG. 29B), a low grade human lipoma (FIG. 29C), ahigh grade human lipoma (FIG. 29D), a myxoid liposarcoma (FIG. 29E) anda dedifferentiated liposarcoma (FIG. 29F). For each tumor sample,prohibitin expression was also evaluated in a control organ from thesame patient (data not shown) to verify that prohibitin was specificallydownregulated in the vasculature of the tumor. FIG. 29 shows thatprohibitin expression is progressively lost in the blood vessels of fattissue, parallel to fat transformation into malignant liposarcomatissues. Thus, prohibitin is a negative indicator of malignancy inadipose tissues, as prohibitin expression is inversely correlated withthe degree of malignancy of the tissue.

A model for prohibitin function in fat vasculature is presented in FIG.31. The KARGG (SEQ ID NO:82) motif, found in reverse orientation in theprohibitin binding peptide CKGGRAKDC (SEQ ID NO:81), shows homology withthe human stem cell growth factor (SCGF) protein (FIG. 31), a member ofthe C-type lectin superfamily. SCGF in combination with VEGF has beenreported to cause differentiation of CD34(+) progenitor cells intoendothelial cells, with characteristics of vascular endothelium (Gehlinget al., Blood, 95:3106-12, 2000). SCGF expression also appears to beassociated with B lymphopoiesis (Witte et al., Eur. J. Immunol.32:1809-17, 1993). It is proposed that binding of SCGF protein to acomplex of prohibitin and the IgM receptor protein in B lymphocytes maymediate B cell differentiation (FIG. 31). It is further suggested thatbinding of CKGGRAKDC (SEQ ID NO:81) to prohibitin in adipose bloodvessel cells may potentially mimic an endogenous SCGF dependentsignaling pathway, perhaps related to endothelial cell differentiation(FIG. 31).

Example 9 Novel Prostate Tumor Targeting Peptides

DU145 prostate tumor cells were injected subcutaneously into the rightfat pad of nude mice. A large phage library (X₂CX₁₄CX₂) was prepared asdiscussed above and 10⁹ phage were injected into male tumor-bearing nudemice. After 24 hr circulation, tumors were removed and phage recoveredfrom the tumors using the bulk method disclosed above. The recoveredphage were amplified, titered and reinjected into a new set of tumorbearing nude mice. The biopanning protocol was repeated for a total ofthree rounds. Ninety-six phage clones recovered from the third round ofbiopanning were selected for sequencing. Translated sequences wereobtained for 76 of the 96 clones.

Targeting peptides recovered from DU145 xenograftic tumors are listed inTable 10. The primary prostate tumor targeting peptides recovered wereYRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) and LGCMASMLREFEGATHACTQ (SEQ IDNO:84). The numbers in parentheses indicate the number of times the sametargeting peptide sequence was obtained. As indicated in Table 9, theYRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) targeting sequence was recovered in11 out of 76 colonies, while the LGCMASMLREFEGATHACTQ (SEQ ID NO:84)peptide was recovered in 8 out of 76 colonies. No obvious homologieswere observed between the prostate tumor targeting peptides listed inTable 10 and any known protein sequence.

TABLE 10 Prostate Tumor-Targeting PeptidesRecovered From DU145 Xenografts YRCTLNSPFFWEDMTHECHA (11) SEQ ID NO: 83LGCMASMLREFEGATHACTQ (8) SEQ ID NO: 84 RGCTEAAGLVIGITTHQCGN (3)SEQ ID NO: 85 IGCNHPSPLGSTVVPTYCFK (3) SEQ ID NO: 86GTCPRQFFHMQEFWPSDCSR (3) SEQ ID NO: 87 DRCVLVRPEFGRGDARLCHS (2)SEQ ID NO: 88 EGCSDIMNTAAERVTGDCSY (2) SEQ ID NO: 89VFCCGSYCGGVEMLASRCGH (2) SEQ ID NO: 90 RECGRTVHRYPWGSPESCER (2)SEQ ID NO: 91 DACSRFLGERVDATAAGCSR (2) SEQ ID NO: 92GNCMGLQVSELFMGPYKCRQ (2) SEQ ID NO: 93 SRCHALRSQSVSTSAGACIS (1)SEQ ID NO: 94 YSCTRLNGTGLQNPPSACDR (1) SEQ ID NO: 95WVCTSASQDTRLKEPGMCIA (1) SEQ ID NO: 96 MHCTSQTLRGTPSLAPKCSD (1)SEQ ID NO: 97 QHCVKGQFPFRESVTITCNS (1) SEQ ID NO: 98HTCWGARDVAQPSGTVRCLK (1) SEQ ID NO: 99 ARCREDTGFMGLGSANICTD (1)SEQ ID NO: 100 RTCEEVRNRALEELTNFCPY (1) SEQ ID NO: 101RTCQVRSNNISPRMALACVT (1) SEQ ID NO: 102 RSCVNSDTGVLQRGAPSCLF (1)SEQ ID NO: 103 RGCWRDSTAWHVSYPVECLA (1) SEQ ID NO: 104NRCMPGFLDDADSAASPCGS (1) SEQ ID NO: 105 NQCSSLLTYQGWKRTKDCIP (1)SEQ ID NO: 106 NDCSAHAQPGWDEVPPMCNQ (1) SEQ ID NO: 107NNCPVEGSQQNYSGATWCRA (1) SEQ ID NO: 108 TTCNKSMSSQPMRDSRECHR (1)SEQ ID NO: 109 TSCVRTGHDENLLKAAYCSS (1) SEQ ID NO: 110TECRGASSGSVSGAATDCRD (1) SEQ ID NO: 111 TLCPPASMGLGREKPRLCSV (1)SEQ ID NO: 112 TLCRSLEHEVGLFKPRECPF (1) SEQ ID NO: 113LRCPLEVDRPNRDPAFLCSQ (1) SEQ ID NO: 114 LGCNKGRYWLSTRLSVSCAL (1)SEQ ID NO: 115 VACDISAVERLPASARSCKT (1) SEQ ID NO: 116VVCFMERQMGTDVVSPMCVN (1) SEQ ID NO: 117 VECVMASASTDGTAAHPCKP (1)SEQ ID NO: 118 VRCNEAQLQDSGTVPHPCLR (1) SEQ ID NO: 119PNCDLDDIVLNPYTAGPCGT (1) SEQ ID NO: 120 PNCYSGDGEISSHIPVQCLM (1)SEQ ID NO: 121 PGCVVSPFALSAQGTSVCTI (1) SEQ ID NO: 122GDCETNNVTKVGGITRNCVG (1) SEQ ID NO: 123 GYCLTVVGGAVLTIALLCVT (1)SEQ ID NO: 124 GPCAATGVNPGDHGAAVCDQ (1) SEQ ID NO: 125GDCETNNVTKVGGITRNCVG (1) SEQ ID NO: 126 KSCGKYGLIVGQPFAEHCPP (1)SEQ ID NO: 127 KLCYRSSAGSELRPPEKCAY (1) SEQ ID NO: 128KICPVTNMWTTPSWAHKCGM (1) SEQ ID NO: 129

To determine the specificity of the prostate tumor targeting peptides,10⁹ phage carrying the targeting peptide sequences YRCTLNSPFFWEDMTHECHA(SEQ ID NO:83) and LGCMASMLREFEGATHACTQ (SEQ ID NO:84) were injectedinto nude mice bearing DU145 xenografts. After 24 hour circulation,tissue samples were obtained from tumor and control organs (kidney,brain, lung and spleen). Tissue samples were washed immediately in DMAMand fixed in 10% formalin for 48 hours at room temperature. Thinsections were stained for phage using anti-phage antibody (1:500dilution) and detected using the DAKO LSAB+ system. The DU145 tumorshowed very heavy staining with anti-phage antibodies (data not shown).No staining was observed for control kidney, brain or lung tissues (notshown). A low level of anti-phage staining was observed in normal spleentissue (not shown). This may be due to the tendency of spleen tissue totrap phage and other foreign particles in general as part of thereticuloendothelial system. In a separate study, samples of theMDA-MB-435 breast carcinoma showed no apparent localization of phagebearing the YRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) sequence.

Competition studies with the YRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) peptidewere performed to determine whether it could inhibit localization ofphage bearing the same targeting sequence to DU145 tumors. Nude micebearing the prostate tumor xenograft were simultaneously injected with300 μg of synthetic YRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) peptide and 10⁹phage bearing the same targeting peptide sequence. A controltumor-bearing mouse was co-injected with fd insertless phage plussynthetic peptide. After 24 hours of circulation, tumor tissue sampleswere removed, washed, fixed, sectioned and stained as disclosed above.Co-administration of synthetic peptide with the same targeting sequenceinhibited the ability of YRCTLNSPFFWEDMTHECHA (SEQ ID NO:83)-phage tolocalize to prostate carcinoma tissue (not shown). No staining ofprostate carcinoma with control fd-tet phage was observed (not shown)

Receptor Purification

The prostate homing receptor for the B2 clone (YRCTLNSPFFWEDMTHECHA SEQID NO:83) was identified. Nude mice bearing DU145 xenografts wereprepared and tissue samples from tumor, kidney and liver were removed.The tissue samples were immediately washed with PBS and three parts ofhomogenization buffer (PBS, 250 mM sucrose, 1 mM EDTA, proteaseinhibitors) was added to one part of tissue sample (about 4 ml). Thetissue with homogenized with an electric grinder, then furtherhomogenized with a dounce homogenizer. After sonication for 1 min onice, the homogenate was centrifuged at 8000×g for 5 min. The supernatantwas removed and the pellet was analyzed for receptor content.

The YRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) peptide was biotinylated andcoupled to NeutrAvidin beads (Molecular Probes, Eugene, Oreg.) usingstandard methods. About 500 μg of biotinylated peptide was incubatedwith 1 ml of NeutrAvidin beads in binding buffer (0.5 M NaCl in PBS)overnight at 4° C. in a 2 ml column. The column was agitated using arotator. Uncoupled peptides were removed and the beads washed threetimes with binding buffer and protease inhibitors. Approximately 1 mg oftissue extract was added to the biotinylated peptide conjugated beads.The material was resuspended in 2 ml of binding buffer and incubatedovernight at 4° C. on a rotator. The material was centrifuged andsupernatant was removed.

The beads were washed four times with wash buffer (0.1% Triton X-100 inPBS with protease inhibitors) and the bound material eluted with 8 Mguanidine HCl. Eluted proteins were analyzed on a 4-20% SDS-PAGEdenaturing gel. Protein (40 μg) from the tumor and kidney were run ascontrols. Bands that showed apparent enrichment for binding to theYRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) peptide were cut out for proteinsequencing.

MALDI analysis of the excised bands identified HSP90 and an unidentifiedprotein. HSP90 is known to be overexpressed in prostate cancer and to beassociated with MHC-I on the cell surface. It is concluded that theendogenous receptor for the YRCTLNSPFFWEDMTHECHA (SEQ ID NO:83) peptideis HSP90α (GenBank Accession No. NM005348).

Example 10 Novel Ovarian Cancer Targeting Peptides

Background

Carcinomas that arise from the ovarian surface epithelium represent agreat challenge in gynecologic oncology (Rosenthal & Jacobs, SeminOncol, 1998. 25:315-25). Ovarian cancer is the sixth most common cancerin women and the deadliest of all gynecologic malignancies, resulting inabout 14,000 deaths annually in the United States. Although theprognosis of ovarian cancer is influenced by many factors capable ofpredicting clinical outcome, including tumor stage, pathological grade,patient performance status and amount of residual disease followingprimary debulking surgery, the biological aspects of ovarian cancer arenot completely understood, implying that there may be other predictiveindicators that could be used. Tumor markers have the potential tocontribute to cancer screening, diagnosis, monitoring, and prognosis aswell as provide targets for anti-tumor therapy. The most extensivelyresearched tumor marker in ovarian carcinoma is CA125. CA125 levels havebeen used as indicators of treatment response or progression. Inmonitoring response to therapy, CA125 is able to reflect progression orregression in over 90% of patients who had elevated preoperative levels.Still, in respect to persistent disease, CA125 only has an accuracy of60-80% and normal values often do not exclude active disease. Thus, theidentification of additional markers with biological relevance would bedesirable.

Neoplasms of the ovary represent a diverse group. They can be dividedinto four major histological classes based on their origin: coelomicepithelial, germ cell, specialized gonadal-stromal, and non-specificmesenchymal. The neoplasms derived from coelomic epithelium are the mostcommon, comprising over 80% of all ovarian tumors. In becomingneoplastic, the coelomic epithelium exhibits a variety of Müllerian typedifferentiation, such as serous, mucinous, endometroid, and clear cell,which comprise the different histological subtypes.

The molecular and cellular events leading to the development of ovariancancer are not completely understood and it is unclear whether ovariancancer follows a stepwise pattern of progression, as no pre-malignantlesion has yet been identified. One proposed theory is that in earlystages the cancer is confined to small epithelial inclusion cysts in theovary. With time, the tumor penetrates through the surface capsule andmalignant cells enter the peritoneal cavity. Here, exfoliation andimplantation are the primary modes of spread of ovarian cancer. Withinthe peritoneal cavity, the cells follow the natural pattern ofperitoneal fluid circulation, leaving all peritoneal surfaces at riskfor tumor cell implantation. Likewise, ovarian cancer may spread bylymphatic dissemination and less commonly by hematogenous route to areassuch as the liver and lungs.

The standard staging system for ovarian cancer is based on surgicalexploration and clinical examination. Stage I is confined to theovaries; stage II is confined to the pelvis; stage III has spreadthroughout the peritoneal cavity; and stage IV is occult distantmetastasis, including parenchymal liver and lung metastasis. Currently,the most powerful determinant of prognosis in ovarian cancer is theextent to which the tumor has disseminated from the primary site at thetime of diagnosis. If diagnosed and treated while the cancer has notspread outside the ovary, the five-year survival rate is 95%. However,only 25% of all ovarian cancers are found at this early stage due tovague symptomatology and lack of effective screening strategies.Moreover, older women with ovarian cancer tend to have a poorerprognosis than younger ones. The overall primary treatment response rateis 80-90%, however, the clinical complete response rate is only 40-50%and the pathological response rate is even lower, about 20-40%. Thus,even with optimal cytoreduction and chemotherapy, many patients remainat risk for the development of recurrent disease.

Since ovarian malignancy may result in the accumulation of ascites inthe peritoneal cavity that contains tumor cells as well astumor-associated immunoglobulins, probing the antibody repertoire in theascites of ovarian cancer patients may result in the identification ofpeptide epitopes resembling tumor antigens. The identified peptideepitopes would correspond to primary sequences found in tumor antigensor mimetopes of such antigens and could potentially serve as markers forthe ovarian cancer. Such markers may be of use for the detection,diagnosis and/or prognosis of ovarian and/or other cancers of the femalereproductive tract.

The phage display methods disclosed above were used to identify noveltumor markers for ovarian cancer. Random peptide phage library werescreened against IgGs isolated from the ascites of ovarian cancerpatients to enrich for phage that bind to ovarian cancer patient IgGsand identify ovarian cancer peptide epitopes. Biochemical methods areemployed to identify the antigen eliciting the antibody response. Theidentified peptide epitopes and corresponding antigens are tested todetermine whether they are linked to disease progression and survival.To assess the value of each motif and corresponding antigen, bankedascites and serum from ovarian cancer patients are screened by an enzymelinked immunosorbent assay (ELISA) protocol.

Materials and Methods

Experimental samples from patients with ovarian cancer were obtainedfrom the M.D. Anderson Cancer Center Specialized Program of ResearchExcellence (SPORE) Ovarian Tumor Bank. Control serum samples wereobtained from healthy blood-donor age-matched women. Ascites sampleswere collected into sterile containers and subjected to centrifugationto separate the cell free fraction from the cellular fraction. The fluidwas stored in aliquots at −20° C. and the remaining cellular fractionprocessed to purify the cells involved in the immune response as well asovarian cancer tumor cells. All blood samples were allowed to clot atroom temperature and then centrifuged. They were promptly aliquoted andfrozen at −20° C. Protein G beads (Pierce) were used for immunoaffinitypurification of IgG from serum and ascites samples. Archivedparaffin-embedded tissue blocks and slides (malignant and non-malignant)collected from the Department of Pathology at the M.D. Anderson CancerCenter were also utilized.

To identify peptide epitopes specific for the anti-tumor immune responsein ovarian cancer, a two-step screening procedure was followed (FIG.34). The peptide library was initially pre-cleared on IVIg (intravenousimmunoglobulins) to remove non-specific peptides. The pre-clearedpeptide phage library was then incubated with IgGs isolated from theascites of ovarian cancer patients. Phage bound to the ovarian cancerIgGs were recovered, amplified, and precipitated for subsequent roundsof biopanning. Following enrichment of a phage population that bound toovarian cancer patient IgGs, individual phage clones were picked forsequence analysis to evaluate enrichment of the most consistentlybinding peptide sequences. Phage display biopanning was performed asdescribed above.

Once the selection rounds were completed as determined by enrichment ofphage capable of binding cancer patient IgGs over control at least 3fold, sequencing and evaluation of the DNA phage insert were undertaken.To sequence the FUSES ssDNA directly, phage ssDNA was prepared using theStrataClean™ resin (Stratagene). StrataClean bead slurry was placed in amicrocentrifuge tube containing phage. The mixture was vortexed stronglyfor 30 seconds and then incubated at room temperature for one minute.The tubes were centrifuged at 2,000×g for 1 minute. The supernatantcontaining the ssDNA was placed into a fresh microcentrifuge tube. Atotal of 3 extractions were performed. The DNA was then precipitatedwith ethanol and used as a template for the sequencing reactionutilizing the primer. Sequencing was done by the chain terminationmethod on an ABI Prism® 3700 (Applied Biosystems/Hitachi. Thecommercially available computer program, DNA Strider, was used in theanalysis of the sequences.

Analysis of the distribution of inserts from the random peptide libraryused a program based on SAS (version 8; SAS Institute) and Perl (version5.0). The program is a high-throughput pattern recognition software usedto analyze short amino-acid residue sequences. The program conducts anexhaustive amino-acid residue sequence count and keeps track of therelative frequencies of n distinct tripeptide motifs representing allpossible n3 overlapping tripeptide motifs in both directions (n<<n3).Counts were recorded for all interior tripeptide motifs, subject only toreflection and single-voting restrictions. No peptide, in the program,is allowed to contribute more than once for a single tripeptide motif(or a reversed tripeptide motif). Tripeptide motifs were chosen for thephage insert analysis because three amino-acid residues seem to providethe minimal framework for structural formation and protein-proteininteraction. Each phage insert analyzed contained seven amino-acidresidues and contributed to ten potential tripeptide motifs.

The Clustal W software from the European Molecular Biology Laboratorywas adopted to analyze the cyclic phage peptides. Clustal W is a generalpurpose multiple sequence alignment program for DNA or proteins andproduces biologically meaningful multiple sequence alignments ofdivergent sequences. It calculates the best match for the selectedsequences, and lines them up so that the identities, similarities anddifferences can be seen.

Construction and purification of GST-fusion peptides. Peptide codingsequences were amplified using colony PCR with the following forward(5′AGGCTCGAGGATCCTCGGCCGACGGGGCT-3′, SEQ ID NO:130) and reverse(5′-AGGTCTAGAATTCGCCCCAGCGGCCCC-3′, SEQ ID NO:131) primers that containBamHI and EcoRI sites (shown in bold), respectively. The amplifiedsequence, containing the peptide coding sequence, was cloned into theBamHI-EcoRI sites of the GST vector, pGEX-2TK (Amersham/Pharmacia), andautomated sequencing used for verification of positive clones. Positiveclones were transformed into the bacterial expression host strain, BL21(DE3) pLys (Stratagene), by electroporation. Expression of theGST-fusion proteins was induced with 200 μM isopropylthiogalactoside(IPTG). Expression of the GST-fusion constructs was compared touninserted pGEX-2TK vector to select for positive clones that producedthe greatest amount of fusion proteins. GST-fusion proteins wereexpressed from selected clones and affinity purified from bacteriallysates by affinity chromatography to immobilized glutathione usingglutathione Sepharose 4B resin (Amersham/Pharmacia).

Testing of individual phage clones (binding assays using patient derivedor control IgGs). Binding of individual phage clones to cancer patientIgGs was studied by a microtiter assay. Antibodies from the ascites ordonor were purified by standard techniques. The antibodies were used tocoat MaxiSorp 96-well plates (Nalge Nunc International Corporation) at aconcentration of 10-100 μg/ml. Coating of plates was carried out at 4°C. overnight. The plates were blocked with 3% Bovine serumalbumin/phosphate buffered saline (BSA/PBS). For the binding reaction,10⁹ TU of phage was added to the coated and blocked plates. The bindingwas performed at room temperature for 2 hours. After the bindingreaction, the wells were washed four times with 3% BSA/PBS. Addition ofK91Kan bacterial culture and incubation at room temperature for 30 minwere used to rescue bound phage. The bacteria were diluted in 10 ml ofLB culture media supplemented with 0.2 μg/ml tetracycline and incubatedfor another 30 min at room temperature. Serial dilutions of thisbacterial culture were plated on LB plates containing 40 μg/mltetracycline. Plates were incubated at 37° C. overnight before countingcolonies. Binding of control (insertless) phage was also assessed.

Inhibition of binding assays. Both GST fusion proteins and syntheticpeptides corresponding the sequence displayed were used for inhibitorystudies. Inhibition studies were performed in a similar manner as thebinding assays described above with the exception that either GST fusionprotein or synthetic peptide corresponding to the phage clone were addedto the experiment. Where the peptide is mediating the interaction withthe immunoglobulins then an inhibition of phage binding should beobserved in a dose dependent manner versus the synthetic peptide. GSTalone and a control peptide containing unrelated amino acids were testedat identical concentrations.

Purification of peptide specific antibodies and immunohistochemistry. Totest if the antigen is indeed specifically expressed in ovarian cancerand tumor-associated, the specific immunoglobulins capable of reactingwith the identified peptide epitopes were purified andimmunohistochemical staining performed on tissue sections from thepatient in whom the initial screening was performed. GST fusion proteinsmade from inserting recombinant peptide sequences of interest in anexpression vector were coated on MaxiSorp multi-well plates (Nalge NuncInternational Corporation). The plates were incubated with the ascitesfluid from which the peptide was originally isolated. Following awashing procedure to remove unbound IgGs, bound IgGs were eluted with0.1 M glycine buffer, pH2.2, neutralized with 1 M Tris-Cl, pH9.0, anddialyzed in PBS overnight. To concentrate the IgG, centricon-30 columns(Millipore) were used. The purified antibody was coupled to biotinaccording to the manufacturer's instructions (Vector). The biotinylatedantibody was analyzed by SDS-gel electrophoresis. Tumor paraffinsections were deparaffinized in xylene, rehydrated in ethanol, andtreated with an antigen retrieval reagent (DAKO) in 10 mM sodiumcitrate, pH 7.5 in a steam bath. Non-specific sites on the tissue wereblocked by incubating the deparaffinized slide in a casein blockingbuffer. Affinity purified biotinylated ovarian cancer ascites fluid IgGswas applied to the sections. A rinsing step and the addition ofstrepavidin conjugated to horseradish peroxidase followed. Positivestaining cells were visualized by the addition of diamino benzidine andsections with phase contrast microscopy with an Olympus IX70 Invertedmicroscope. All sections were additionally counterstained withhematoxylin.

Protein homology searches. Database searches may be helpful in theidentification of the antigen for a given peptide sequence. Validatedpeptide epitope(s) were searched in online databases (through theNational Center for Biotechnology Information (NCBI;http://www.ncbi.nlm.nih.gov/BLAST/) and candidate tumor antigens wereidentified by homology with known human proteins.

Analysis of Patients Eliciting an Immune Response Against the IdentifiedPeptide(s).

An ELISA protocol was used to examine the presence of antibodies for theselected markers in a panel of ovarian cancer patients. Normal serum andnon-malignant ascites were also tested to help show whether or not theimmune response to the marker was associated with evaluated patientcharacteristics.

ELISA. Peptide sequences of interest were expressed as GST fusionproteins (described above) and used to screen banked ascites and serumto determine the role of the humoral response against these markers. Thepurified GST-fusion proteins were used to coat a 96-well plate at 100ng/well at room temperature (RT) or at 4° C. overnight. Followingcoating, the wells were emptied, rinsed, and non-specific sites wereblocked with 200 μl 3% BSA/PBS at RT for 1-2 hours. Cancer patientascites and/or sera were applied to each coated and blocked well at1:100 dilution and then incubated at RT for 1 hour. The wells wererinsed 3× with 3% BSA/PBS containing 0.01% Tween 20, and then incubatedfor 1 hour with 50 μl each of anti-human alkaline phosphatase at 1:2000dilution. Signals were detected in the presence of p-nitrophenylphosphate by measuring OD₄₀₅ at specific intervals to follow the courseof color development. A positive control was the cancer ascites thepeptide was identified from, and a negative control was donor seraand/or BSA.

Results

FIG. 35 shows the results of biopanning a CX₇C phage display libraryagainst ascites taken from an ovarian cancer patient after 2 and 3rounds of biopanning. As can be seen, after two rounds of biopanning thetargeting phage specificity was fairly low, exhibiting higher levels ofbinding to the BSA and control immunoglobulins. However, after a thirdround of biopanning the phage exhibited a very high degree ofselectivity for binding to the ovarian cancer patient's immunoglobulins,compared to control IgGs or BSA.

The primary peptide sequence recovered against ovarian cancer patientascites exhibited the targeting sequence CVPELGHEC (SEQ ID NO:132). Thispeptide represented 86% (73 of 85) of the phage clones that weresequenced. Additional studies were carried out to validate the ovarianascites targeting specificity of this peptide sequence. FIG. 36 showsthat antibodies isolated from the ascites of an ovarian cancer patientbound specifically to the targeting peptide sequence CVPELGHEC (SEQ IDNO:132). Purified ascites immunoglobulins were exposed to microtiterplates containing immobilized GST-CVPELGHEC (SEQ ID NO:132) fusionproteins. Antibody binding to the immobilized fusion protein wascompetitively inhibited in a dose-dependent fashion by the syntheticCVPELGHEC (SEQ ID NO:132) peptide, but was unaffected by a controlpeptide (FIG. 36).

Ascites from patients with different stages of ovarian cancer,non-ovarian cancer or non-malignant conditions was screened againstGST-CVPELGHEC (SEQ ID NO:132) fusion proteins using serial dilutions todetermine the optimal reactivity of immunoglobulins present in eachsample. The results, presented in FIG. 37, show that peptide binding toimmunoglobulins is stage dependent, with ascites from Stage 1V ovariancancers showing a higher reactivity than ascites from Stage III ovariancancer. Some reactivity was also observed with ascites from non-ovariancancer, but not with ascites from patients with non-malignant conditions(FIG. 37).

These results demonstrate the utility of the CVPELGHEC (SEQ ID NO:132)peptide for the detection, diagnosis, staging and/or prognosis ofovarian cancer. The present of antibodies reactive with the CVPELGHEC(SEQ ID NO:132) peptide in ascites from suspected ovarian cancerpatients is indicative of the presence of a high stage ovarian cancer.The skilled artisan will realize that the presence of anti-CVPELGHEC(SEQ ID NO:132) antibodies in patient ascites may also be indicative ofthe presence of non-ovarian cancers. The artisan will further realizethat the CVPELGHEC (SEQ ID NO:132) peptide may be of use as a mimeotopeof an ovarian cancer selective endogenous protein. As discussed above,the endogenous mimeotope of the CVPELGHEC (SEQ ID NO:132) peptide may beidentified by protein homology searches of the CVPELGHEC (SEQ ID NO:132)peptide against standard databases. Alternatively, as disclosed above,antibodies binding to the CVPELGHEC (SEQ ID NO:132) peptide may bepurified by immunoaffinity chromatography and used to identify theendogenous mimeotope. Also alternatively, monoclonal antibodies reactivewith the CVPELGHEC (SEQ ID NO:132) peptide may be prepared by standardmethods and used to identify the endogenous mimeotope. A preliminaryBLAST search against the NCBI database did not reveal any obvioushomologies with known protein sequences, indicating that the ovariancancer targeting peptide may mimic an epitope comprised of two or moreportions of the primary sequence of the endogenous mimeotope.

Immunohistochemical analysis against ovarian cancer thin sections fromthe same patient whose ascites was screened for reactive antibodiesdemonstrated that an endogenous mimeotope was in fact present in theovarian tumor (not shown). Autologous immunopurified immunoglobulinsused for IHC versus a primary ovarian lesion as well as a metastaticperitoneal nodule showed the presence of strong immunoreactive staining(not shown). Negative controls using secondary antibody alone, or incombination with immunoglobulins obtained from a pool of non-cancerpatients showed no IHC staining under identical conditions (not shown).A recombinant GST-CVPELGHEC (SEQ ID NO:132) fusion protein inhibitedstaining with autologous immunoglobulins. These results demonstrate thatIgGs from the ascites of ovarian cancer patients are reactive against anendogenous ovarian cancer antigen that is of use for ovarian cancerdetection, diagnosis and/or staging.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it areapparent to those of skill in the art that variations maybe applied tothe COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the methods described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itare apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-57. (canceled)
 58. A method comprising steps of: administering to asubject in need thereof a composition comprising: a targeting moietycharacterized by selective binding to GRP78; and an imaging agentattached to the targeting moiety.
 59. A method comprising steps of:administering to a subject in need thereof a composition comprising: atargeting moiety characterized by selective binding to IL-11Ra; and animaging agent attached to the targeting moiety.